0042~6989/91 $3.00+ 0.00 Cop*ght 0 1991Pergamon Press plc
YisionRes. Vol. 31, No. 7/8, pp. IID-1127, 1991 Printed in Great Britain. All rights reserved
MUSCARINIC ACETYLCHOLINE RECEPTOR-MEDIATED PHOSPHOINOSITIDE TURNOVER IN CULTURED HUMAN RETINAL PIGMENT EPITHELIUM CELLS N. N. OSBORNE,F. FITZGIB~~ and G. SCHWARTZ Nufbeld Laboratory of ~htha~alo~,
University of Oxford, Walton Street, Oxford OX2 6AW, U.K.
Ahatraet-Cultured retinal pigment epithelium cells prepared from post-mortem adult human eyes are shown to contain muscarinic receptors associated with phosphoinositide turnover. Carbachol at a ~on~nt~tion of 100 p M induced a four-fold increase in ‘H-inositol phosp~t~ (more than 74% is in the form of -‘H-inositol-l-phosphate) accumulation within 45 min in cells prelabelled with ‘H-myoinositol and exposed to 5 mM LiCl. The PCs0 of carbachol was approx. 70 PM and the saturation concentration was about 1 mM. The carbachol-induced response was blocked by both atropine and pirenxepine, the former being most effective. Pm-exposure of c&s to carbachol resulted in desensitization and a drastic reduction in the subsequent ~~bachol-ind~d stimulation of ‘H-inositol phosphates. The carbachol response could be at~nuated by the biolo~~~ly active phorboi ester, 4~-phor~l 1Zmyristate 13-acetate, and this was nullified by the protein kinase C inhibitor, staurosporine. The biolo~~ly inactive phorbol ester, 4a-phorbol 12,I3 dideconoate, did not attenuate the carbachol-induced stimulation of 3H-inositol phosphates, Pertussis toxin failed to inflnence the carbachol receptor-mediated phosphoinositide turnover. These studies provide clear evidence for the occurrence of muscarinic receptors coupled to phosphoinositide hydrolysis on human retinal pigment epithelium cells. Phosphoinositide
turnover
Pigment ~itheli~
cells
Activation ofmuscarinic acetylcholine receptors leads to at least two distinct biochemical events: the inhibition of adenylate cyclase activity and the stimulation of phosphoinositide-specific phospholipase C (Brown & Brown, 1984; Brown & Goldstein, 1986; Akiyama, Vickroy, Watson, Roeske, Reisine, Smith & Yamamura, 1986). Receptor-coupled activation of phospholipase C is also an event induced by many other neurotransmitters, neurom~~ators and hormones which use calcium as a mes~nger (Downes, 1986; Osborne, Tobin 62 Ghazi, 1988a). The key reaction of this transducing system is the hydrolysis of phosphatidylinositol-4,5-bisphosphate to form two metabolites, diacylglycerol and inositol triphosphate, both of which may serve as a second messenger to trigger the signal cascade. Diacylglycerol stimulates a calcium and phospholipid-dependent protein kinase C, whereas inositol triphosphate acts by mobilising intracelltdar calcium. The retinal pigment epithelium (RPE) is a monolayer of cells located on Bruch’s membrane
Cholinergic receptors
between the retinal photoreceptor and the cho~o~apilla~es. It performs a variety of functions which are necessary for the maintenance of photoreceptor physiology, such as regulation of the transport nutrients, viz. vitamin A, and other materials between the choroid and photoreceptors. Several types of receptor have been demonstrated in chick RPE cells linked to the production of the secondary messenger c-AMP (Koh 8z Chader, 1984a, b). In addition j?-receptors, which are also associated with c-AMP metabohsm, have been shown to exist on human RPE cells {F~edman, Hackett & Campochiaro, 1987). To date, receptor-coupled activation of phospholipase C for acetylcholine or, indeed, for any other agonist, has not been demonstrated in RPE cells of any species, although the recent report by Friedman, Hackett and Campochiaro (1988) showed that acetylcholine and carbachol cause an increase in intracellular calcium concentrations of cultured RPE cells, thus suggesting the occurrence of such receptors. The present study provides proof of the presence of mu~a~nic receptors coupled to &heactivation of phospholipase C in human RPE cells.
1119
N.N.
1120 MATERIALS
OSBORNE
AND METHODS
Methods Preparation of primary cultures of human RPE. Post-morten adult eyes (received l-2 days
following death) were stored overnight at 4°C and the transferred to a sterile cabinet. The cornea just below the ora serrata was dissected and the lens, vitreous and retina carefully removed. The eye was carefully rinsed with about 500 ~1 PBSA solution (137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH,P0,*H20, 7 mM Na,HPO,, pH 8.2). A 0.25% trypsin solution (in PBSA) was then added to the eye cup (to below the cut edges to avoid contamination) and incubated for 60 min at 37°C in a moist 5% CO2 atmosphere. Most of the trypsin solution was carefully removed and replaced with a culture solution (120 ml distilled water, 20 ml foetal calf serum-heat inactivated, 10 ml 285 mM sodium bicarbonate solution, 20 ml 10 x Hams FlO solution, 2 ml 200 mM glutamine, 6 ml 0.44 M glucose, 2 ml of 10 mg/ml gentamycin, pH 7.4). With a brush the RPE cells were suspended in the culture solution, transferred to a tube and centrifuged at 70g for 7 min at 4°C. The pellet was resuspended in a fresh culture solution and cells were grown in 6 ml Hams FlO medium in 25 cm* tissue culture flasks (from Sterilin) to passage four. Cells (from passages 2-4) were grown on 13 mm diameter glass cover-slips [previously coated with a solution (10 ,ug/ml) of poly l+lysine] in Multidish (Falcon 3008) wells at a density of 5-10 x lo4 cells per coverslip. The cultures were grown at 35.5”C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was changed at 3-day intervals. Stimulation of inositol phosphates
(InsPs).
Coverslips containing confluent cultures (4-6 days subsequent to plating) were incubated for 15 hr at 35.5”C (in a 5% CO? humidified atmosphere) in Eagles Minimal Essential medium (MEM) plus glucose (final concentration 33 mM) containing 0.1 PM ‘H-myoinositol. An incubation time of 15 hr was routinely used since trial experiments showed that at this time isotopic equilibrium was maximal. The cultures were then rinsed with MEM solution containing 5 mM LiCl (lithium/MEM solution) and then preincubated in the same solution for 15 min. Each culture was then transferred to another Falcon multiwell which contained 290 ~1 lithium/MEM solution plus a further 10 ~1 of this solution either alone or containing an
et al.
antagonist. The cultures were then incubated for 10min at 35.5”C, after which a further 10~1 lithium/MEM solution containing agonist, was added. Control cultures were simply supplied with vehicle solution. Each coverslip containing a culture was therefore incubated with a total volume of 310 ~1 of solution. After an incubation period of 45 min at 35.5”C the reaction was stopped by transferring cultures on two or three coverslips into 1 ml chloroform methanol (1: 2, v/v) present in a glass tube. Subsequently, the media from two or three wells plus an equal volume of chloroform were added to each glass tube. The coverslips and RPE cells were crushed with a glass rod inside the glass tubes, vortexed, and then left at 5°C overnight to allow the complete separation of the aqueous phase from the lower liquid phase. The water soluble 3H-InsPs were then extracted using Dowex as described by Berridge, Downes and Hanley (1982). Materials
Myo-(2-3H)-inositol, 12 Ci/mmol was from Amersham International, U.K. Foetal calf serum, 10 x Hams FlO, MEM and glutamine was brought from GIBCO, U.K. Gentamycin, pertussis toxin and phorbol esters were from Sigma, U.K. All other chemicals were purchased from Sigma or from BDH (U.K.). RESULTS
The identity of the human RPE cultures from a 4th passage 6 days after being plated and “stained” immunohistochemically for the localization of vimentin-immunoreactivity is shown in Fig. 1 (see Osborne, Morgan, Ghazi & Beaton, 1988b for details of methodology and source of antibody). The cells appear homogenous in nature and are confluent. (a) E#ect of carbachol on the accumulation of ‘H-InsPs
Addition of carbachol to the RPE culture induced a dose-dependent increase in ‘H-InsPs accumulation in the presence of 5 mM LiCl (Fig. 2). Carbachol at a concentration as low as 10 fl M evoked a 110% increase in InsPs accumulation within 45 min of incubation, and, at the saturation concentration (1 mM), the increase was 590% relative to basal level. From the dose-response relationship, the ECSo value of carbachol was found to be about 70 p M.
Fig. I. Immunohistochemical localization of vimentin-like immunoreactivity in a 20 day-old human pigment epithelium culture. It can be seen that the cells are confluent at this stage in the culture. Scale bar 50pM.
1121
Phosphoinositide Dose
response
curve
turnover in human RPE cells
1123
RPE
for
5 ii
500 250
E’
i Corbachol concentration
(@A)
Fig. 2. Concentration-response relationship of carbacholinduced 3H-InsPs accumulation in pigment epithelium cells. The cells were labelled overnight for 15 hr with 3H-myoinositol and then washed to remove the isotope. After preincubation with 5 mM LiCl at 355°C for 15 min, various concentrations of carbachol were added and the cells were further incubated for 45 min. The accumulation of ‘H-InsPs was then measured using Dowex as described by Berridge et al. (1982). The data presented are mean + SEM of three independent experiments which were performed in triplicate. The basal activity in the absence of carbachol was expressed as 100% and was 3 15 k 100 dpm per sample.
The carbachol-induced InsPs accumulation was linearly increased for the first 45 min of incubation and continued to increase in a non-linear fashion up to 120 min (Fig. 3); at the latter time, a 610% stimulation was observed. Separation of ‘H-Ins- l-P, 3H-Ins-2-P and 3H-Ins-3-P following the method of Berridge et al. (1982) showed that most of the radioactivity was associated with ‘H-Ins- 1-P after 45 min stimulation by carbachol (see Fig. 4). This was also the case after 15 min stimulation with carbachol, whereas after a period of only 5 min the radioactivity was not so unequally distributed between the different 3H-InsPs. Effect 700
of
IOOpM
corbachol
r-
5 mill
15min
45 mln
Fig. 4. Comparative distribution of 3H-Ins-l-P, ‘H-Ins-2-P and ‘H-Ins-3-P following stimulation of pigment epithelium cells for 5, 15 and 45 min with 100 PM carbachol. The methods used were as described in the text and Fig. 1, except that the accumulated )H-InsPs was eluted from the Dowex column by stepwise elution, using different concentrations of ammonium formate and formic acid as described by Berridge et al. (1982). Results are mean + SEM of three independent experiments.
Atropine, a nonselective muscarinic antagonist and pirenzepine, a putative M, muscarinic receptor antagonist were assessed for their ability to block the increased accumulation of InsPs evoked by carbachol. Table 1 shows that while both atropine and pirenzepine do antagonize the effect of carbachol, atropine is the most effective. Prazosin, the a,-adrenergic receptor antagonist and ketanserin, the 5-HT, receptor blocker failed to influence the carbacholinduced response at concentrations as great as 10pM. (b) Desensitization of carbachol-mediated phosphoinositide turnover
In order to assess the desensitization of carbachol-induced accumulation of InsPs, cultures were pretreated with 100 PM carbachol throughout the period of incubation with radioactive inositol. Thereafter the cells were washed
T
Table 1. Effect of various antagonists on the carbachol-induced stimulation of ‘HInsPs accumulation in pigment epithelium cells. The concentration of carbachol was 1OOpM. Results are expressed in terms of percentage inhibition and are the means of three separate experiments each carried out in triplicate
I 0
I
I
15
30
1 45
1
1
I
I
’
60
75
90
105
120
Time (mh)
Fig. 3. Time course of carbachol-induced 3H-InsPs accumulation in pigment epithelium cells. Experimental conditions were described in the text, except that cells were incubated for various periods of time in the presence of 100 PM carbachol. The data presented are mean + SEM of a triplicate experiment, repeated three times.
Antagonist Atropine (1 PM) Atropine (5 FM) Pirenzepine (5 p M) Pirenxepine (10 PM) Ketanserin (10 PM) Prazosin (10 bM)
% Inhibition of carbachol response 62 k 9 78 + 7 31+9 48 k 7 10 f 4 8z%4
N.B. Ketanserin and prazosin effects were not significant.
1124
N.N. OSBORNEet al.
laJ n
n-
2000
[]
Basal
"6
1750
•
Stimulated
._~
1500
H ~
1250
~ ~ 400
1000 ~,~
700
~
~
300
5 20o
500
N ~
o
Control
P re-- exposure to CCh
[ ] I/~M ÷ Stourosporine [ ] d/zg/ml
500
750
250
[ ] Control I&-~OA/~M • I/~M
100
1iiniiD1i PMA
Inactive Phorbol Ester
PTX
Fig. 5. Carbachol-induced 3H-InsPs accumulation in retinal pigment epithelium cells pre-exposed to muscarinic receptor agonist carbachol. Cells were incubated in the absence or presence of carbachol (100#M) for 15hr during the cell labelling and then washed three times before challenging them with 100 p M carbachol (stimulated values). It can be seen that while the basal level of 3H-InsPs was not influenced by pre-exposure to carbachol, the stimulated value was much reduced. Results are mean ___SEM of three independent experiments.
Fig. 6. Effect of phorbol esters and pertussis toxin on carbachol-induced stimulation of 3H-InsPs accumulation. Labelled cells were exposed to phorbol esters 15 min before being challenged by the addition of 100 #M carbachol. In the experiments with staurosporine, the substance was added 5 min before the addition of phorbol esters. In the case of the pertussis toxin (PTX) experiment, the substance was added to the culture during the 15 hr incubation with 3H-myoinositol. Results are mean values __+SEM for 3~J, separate experiments.
free o f c a r b a c h o l a n d r a d i o a c t i v i t y a n d then challenged with c a r b a c h o l in the n o r m a l w a y in the presence o f LiC1. A s s h o w n in Fig. 5, p r e - e x p o s u r e to c a r b a c h o l causes a m a r k e d i n h i b i t i o n o f 3H-InsPs a c c u m u l a t i o n i n d u c e d b y s u b s e q u e n t s t i m u l a t i o n with 100 # M c a r b a c h o l . Basal values for 3H-InsPs were n o t d r a s t i c a l l y affected b y p r e - e x p o s u r e to c a r b a c h o l . T h e results t h e r e f o r e suggest t h a t p r e - e x p o s u r e to c a r b a c h o l causes a d e s e n s i t i z a t i o n o f the receptors.
are m i l d e r a n d p i l o c a r p i n e is the least effective. T h e influence o f s e r o t o n i n a n d q u i s q u a l a t e was negligible w h e r e a s n o r a d r e n a l i n e s t i m u l a t e d 3HInsPs, b u t m u c h less effectively t h a n c a r b a c h o l .
(c) Comparison of the effect of carbaehol and other agonists on phosphoinositide turnover T a b l e 2 shows the c o m p a r a t i v e effects o f 100 # M o f v a r i o u s cholinergic agonists ( c a r b a chol, p i l o c a r p i n e , m u s c a r i n e a n d arecoline), n o r a d r e n a l i n e , s e r o t o n i n a n d q u i s q u a l a t e on the s t i m u l a t i o n o f 3H-InsPs a c c u m u l a t i o n in R P E cultures. T h e m o s t effective substance is c a r b a chol. A l t h o u g h the o t h e r cholinergic substances s t i m u l a t e 3H-InsPs a c c u m u l a t i o n , their effects Table 2. Effects of various agonists on 3H-InsPs accumulation in pigment epithelium cells. Results are expressed in terms of percentage accumulation above basal levels. Results are mean values __+SEM where n = 4 Substance (I00 pM) Carbachol Pilocarpine Muscarine Arecoline Noradrenaline Serotonin Quisqualate
% Stimulation of 3H-InsPs 408 _+32 94 __+28 226 + 55 154 + 39 84 _+22 29 4- 18 33 _+ 19
(d) Effect of phorbol esters and pertussis toxin T h e p r o t e i n kinase C a c t i v a t o r , P M A (4//p h o r b o l 12-myrisate 13-acetate) a n d the biologically inactive p h o r b o l ester, 4 ~ - p h o r b o l 12,13 d i d e c o n o a t e were e x a m i n e d for their effects on the c a r b a c h o l - i n d u c e d s t i m u l a t i o n o f 3H-InsPs a c c u m u l a t i o n . P M A at c o n c e n t r a t i o n s o f 0.1 a n d 1/~M i n h i b i t e d 3H-InsPs a c c u m u l a t i o n b y 35% a n d 4 6 % respectively w h e r e a s the inactive p h o r b o l ester failed to a t t e n u a t e the response in the s a m e c o n c e n t r a t i o n r a n g e (Fig. 6). A t a c o n c e n t r a t i o n o f 1 # M s t a u r o sporine, a p r o t e i n kinase C i n h i b i t o r (see H i d a k a & H a g i w a r a 1987; H u a n g 1989), nullified the effect p r o d u c e d by P M A . Pertussis toxin is k n o w n to i n a c t i v a t e Gi p r o t e i n t h r o u g h A D P r i b o s y l a t i o n o f the alphai s u b u n i t ( K a t a d a & U i 1982). The a d d i t i o n o f pertussis toxin (1 # g per ml) to the r a d i o a c t i v e inositol over a p e r i o d o f 15 hr did n o t affect the extent o f 3H-InsPs a c c u m u l a t i o n i n d u c e d b y c a r b a c h o l : basal values were n o t significantly affected by pertussis toxin either. DISCUSSION These results p r o v i d e the first p r o o f o f the presence in c u l t u r e d h u m a n R P E cells o f m u s c a r i n i c r e c e p t o r s linked to p h o s p h o i n o s i t i d e turnover. T h e ECs0 value o f c a r b a c h o l for
Phosphoinositide
turnover in human RPE cells
InsPs accumulation is lithium-dependent about 70pM and maximum stimulation for 100 PM carbachol reached 6-fold after about lOOmin. This agonist potency and the degree of stimulation are similar to what is found in other systems, including retinal slices (Osborne, 1988; Cutcliffe & Osborne, 1987), brain slices (Batty & Nahorski, 1985), astrocytes (Pearce, Cambray-Deakin, Marrow, Grimble & Murphy, 1985) and NCB-20 cells (Chuang, 1986). The carbachol-induced response was inhibited by two muscarinic antagonists, atropine and pirenzepine, the latter being weaker. This suggests that the carbachol-induced phosphoinositide turn-over might be mediated in human RPE cells through Ml-type muscarinic receptors. This would be consistent with the observation made by Friedman et al. (1988) that stimulation of muscarinic receptors in human RPE leads to a mobilization of internal calcium levels, an observation most often associated with pharmacologically characterized M,-type receptors. In addition, receptors mediating an increased accumulation of InsPs would be expected to result in a simultaneous increase in the mobilization of internal calcium levels caused specifically by the action of Ins-3-P (see Berridge & Irvine, 1989; Osborne & Ghazi, 1990). Of the muscarinic agonists tested, carbachol was the most effective in stimulating InsPs accumulation in human RPE cultures. The order of effectiveness for the various muscarinic agents is similar to what has been described for the stimulation of InsPs in retinal slices (Osborne, 1988) and may be explained by the fact that the agents used range from being partial agonists (e.g. pilocarpine) to full agonists (e.g. carbachol). The specificity of the cholinergic receptors is exemplified by the finding that other antagonists of receptors linked to stimulation of phosphoinositide turnover, viz. ketanserin (5-HT, receptor antagonist) and prazosin (a,-receptor agonist) were ineffective in blocking the carbachol effect at concentrations as great as 10 v M. In comparison with carbachol, other potential agonists such as noradrenaline (a,-receptors), serotonin (5-HTz receptors) and quisqualate (quisqualate receptors) hardly stimulated phosphoinositide turnover (see Table 2). This differs from what occurs in the retina, where the order of effectiveness in stimulating InsPs is quisqualate > carbachol > noradrenaline > serotonin (see Osborne & Ghazi, 1990; Osborne, 1990; Ghazi & Osborne,
1125
1988). The present results therefore show that @,-type, 5-HT,-type and quisqualate-type receptors linked to InsPs production are either absent or present in low amounts in human RPE cells. Further experiments are necessary to clarify this point. The carbachol-induced response in the RPE cells could be desensitised by pretreatment with carbachol. This is not a surprising finding, although it is not known by which mechanism this occurs. It is conceivable that the continuous accumulation of diacylglycerol and InsPs results in a persistent activation of muscarinic receptors, which leads to an activation of protein kinase C and phosphorylation of specific proteins. The protein phosphorylation may trigger down-regulation of muscarinic receptor binding sites, or alternatively, an internalization of receptors which could be associated with the normal recycling process of receptors. The biologically active phorbol ester, PMA, was able to attenuate carbachol-induced InsPs accumulation, whereas the biologically inactive phorbol ester, 4a -phorbol 12,13-didecoate, was ineffective in causing the effect in RPE cells. The PMA effect was nullified by the PKC inhibitor, staurosporine. These data are analogous with results obtained by various investigators using other receptor systems (Ghazi & Osborne, 1989; Roth, Nakaki, Chuang & Costa, 1986; Chuang 1986; Labarca, Janowsky, Pate1 & Paul, 1984). It is well established that active phorbol esters act on protein kinase C to induce their effects (Nishizuka, 1984). The inhibitory effect produced by PMA shows that protein kinase C activation alters muscarinic receptor-mediated responses in human RPE. Likely sites of action of activated protein kinase C include: (a) the receptor, (b) GTP binding protein, (c) phospholipase C or (d) indirectly through, e.g. c-AMP 2nd messenger systems. Further experiments are clearly necessary to discover whether any of these mechanisms are involved. Irrespective of the process, the present results suggest that diacylglycerol formation during physiological muscarinic-induced activation in RPE cells may have a negative feedback on the production of InsPs via action of protein kinase C. Such a process would help maintain the cellular physiological homeostasis. As in other systems (Schimmel & Elliott, 1986; Uhing, Prpic, Jiang & Exton, 1986), pretreatment with pertussis toxin for as long as 15 hr did not affect basal or carbacholinduced InsPs accumulation in RPE cells.
N. N. OSBORNE et al.
1126
Under similar conditions, the carbacholinduced attenuation of adenylate cyclase activity was reduced by more than 50% in iris/ ciliary muscle (Tobin & Osborne, 1988). The lack of effect of pertussis toxin in the muscarinic receptor-mediated InsPs accumulation in RPE cells is in contrast to the inhibition by pertussis toxin of receptor phosphoinositide turnover in some other systems (Brass, Laposta, Banga & Rittenhouse, 1986; Pfeilschifter & Bauer, 1986) and suggests that the GTP regulating protein of the MI-type muscarinic receptors in RPE cells may be distinct from the Gi protein. In conclusion, the present results show that muscarinic receptors linked to InsPs accumulation exist in human RPE cells. It remains to be determined exactly where these receptors are local&d on the RPE cells in order to determine their physiological relevance. Should the receptors be exclusively associated with the apical surfaces of the RPE cells then consideration has to be given to the possibility that the photoreceptors provide the source of the natural agonist (viz. acetylcholine). Present evidence suggests, however, that photoreceptors of mammals are not choline@, though it is known that in lower vertebrates, such as mudpuppy and turtle, certain photoreceptors may synthesise acetylcholine (see Pure, 1985). Acknowledgements-We
are extremely grateful to the British Retinitis Pigmentosa Society and the Royal National Institute for the Blind for financial support.
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Nishizuka, Y. (1984). The role of protein kinase C in cell surface receptor signal transduction and tumour promotion. Nature, London, 308, 693698. Osborne, N. N. (1988). Muscarinic stimulation of inositol phosphate formation in rat retina: Developmental changes. Vision Research, 8, 875-888. Osborne, N. N. (1990). Stimulatory and inhibitory actions of excitatory amino acids on inositol phospholipid metabolism in rabbit retina: Evidence for specific quisqualate
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Osborne, N. N. & Ghazi, H. (1990). Agonist-stimulated inositol phospholipid hydrolysis in the mammalian retina. In: Osborne, N. N. & Chader, G. (Eds.), Progress in retinal research. Vol. 9, pp. 101-134. Oxford: Pergamon Press. Osborne, N. N., Tobin, A. B. & Ghazi, H. (1988a). Role of inositol triphosphate as a second messenger in signal transduction processes: An essay. Neurochemistry Research, 13, 177-191. Osborne, N. N., Morgan, B., Ghazi, H. & Beaton, D. W. (1988b). Correlation between vimentin, GABA and GFAP immunoreactivities and 3H-GABA and 3H-taurine uptake in intact rabbit retina and retinal cell cultures. Biogenic Amines, 5, 249-268.
Pearce, B., Cambray-De&in, M., Marrow, C., Grimble, J. & Murphy, S. (1985). Activation of muscarinic and a,-adrenergic receptors on astrocytes results in the accumulation of inositol phosphates. Journal of Neurochemistry, 45, 1534-1540. Pfeilschifier, J. & Bauer, C. (1986). Pertussis toxin abolishes angiotensin II-induced phosphoinositide hydrolysis and
1127
synthesis in rat renal mesangial cells.
Puro, D. G. (1985). Cholinergic systems. In: Morgan, W. W. (Ed.), Retinal transmitters and modulators for the brain. (Vol. 1, pp. 65-91). FL: CRC Press. Roth, B. L., Nakaki, T., Chuang, D. M. & Costa, E. (1986). 5-HT, receptors coupled to phospholipase C in rat aorta: Modulation of phosphoinositide turnover by phorbol ester. Journal of Pharmacology and Experimental Therapeutics, 238, 480-485.
Schimmel, R. J. & Elliott, M. E. (1986). Pertussis toxin does not prevent alpha adrenergic stimulated breakdown of phosphoinositides or respiration in brown adipocytes. Biochemical and Biophysical Research Communicarions, 135, 823-829. Tobin, A. B. & Osborne, N. N. (1988). Evidence for the presence of choline+ muscarinic receptors negatively linked to adenylate cyclase in the iris/ciliary body. Neurochemisrry Intemarional, 13, 517-523.
Uhing, R. J., Prpic, V., Jiang, H. UCExton, J. H. (1986). Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. Journal of Biological Chemistry, 261, 2140-2146.
YisionRes. Vol. 31, No. 7/8, pp. IID-1127, 1991 Printed in Great Britain. All rights reserved
MUSCARINIC ACETYLCHOLINE RECEPTOR-MEDIATED PHOSPHOINOSITIDE TURNOVER IN CULTURED HUMAN RETINAL PIGMENT EPITHELIUM CELLS N. N. OSBORNE,F. FITZGIB~~ and G. SCHWARTZ Nufbeld Laboratory of ~htha~alo~,
University of Oxford, Walton Street, Oxford OX2 6AW, U.K.
Ahatraet-Cultured retinal pigment epithelium cells prepared from post-mortem adult human eyes are shown to contain muscarinic receptors associated with phosphoinositide turnover. Carbachol at a ~on~nt~tion of 100 p M induced a four-fold increase in ‘H-inositol phosp~t~ (more than 74% is in the form of -‘H-inositol-l-phosphate) accumulation within 45 min in cells prelabelled with ‘H-myoinositol and exposed to 5 mM LiCl. The PCs0 of carbachol was approx. 70 PM and the saturation concentration was about 1 mM. The carbachol-induced response was blocked by both atropine and pirenxepine, the former being most effective. Pm-exposure of c&s to carbachol resulted in desensitization and a drastic reduction in the subsequent ~~bachol-ind~d stimulation of ‘H-inositol phosphates. The carbachol response could be at~nuated by the biolo~~~ly active phorboi ester, 4~-phor~l 1Zmyristate 13-acetate, and this was nullified by the protein kinase C inhibitor, staurosporine. The biolo~~ly inactive phorbol ester, 4a-phorbol 12,I3 dideconoate, did not attenuate the carbachol-induced stimulation of 3H-inositol phosphates, Pertussis toxin failed to inflnence the carbachol receptor-mediated phosphoinositide turnover. These studies provide clear evidence for the occurrence of muscarinic receptors coupled to phosphoinositide hydrolysis on human retinal pigment epithelium cells. Phosphoinositide
turnover
Pigment ~itheli~
cells
Activation ofmuscarinic acetylcholine receptors leads to at least two distinct biochemical events: the inhibition of adenylate cyclase activity and the stimulation of phosphoinositide-specific phospholipase C (Brown & Brown, 1984; Brown & Goldstein, 1986; Akiyama, Vickroy, Watson, Roeske, Reisine, Smith & Yamamura, 1986). Receptor-coupled activation of phospholipase C is also an event induced by many other neurotransmitters, neurom~~ators and hormones which use calcium as a mes~nger (Downes, 1986; Osborne, Tobin 62 Ghazi, 1988a). The key reaction of this transducing system is the hydrolysis of phosphatidylinositol-4,5-bisphosphate to form two metabolites, diacylglycerol and inositol triphosphate, both of which may serve as a second messenger to trigger the signal cascade. Diacylglycerol stimulates a calcium and phospholipid-dependent protein kinase C, whereas inositol triphosphate acts by mobilising intracelltdar calcium. The retinal pigment epithelium (RPE) is a monolayer of cells located on Bruch’s membrane
Cholinergic receptors
between the retinal photoreceptor and the cho~o~apilla~es. It performs a variety of functions which are necessary for the maintenance of photoreceptor physiology, such as regulation of the transport nutrients, viz. vitamin A, and other materials between the choroid and photoreceptors. Several types of receptor have been demonstrated in chick RPE cells linked to the production of the secondary messenger c-AMP (Koh 8z Chader, 1984a, b). In addition j?-receptors, which are also associated with c-AMP metabohsm, have been shown to exist on human RPE cells {F~edman, Hackett & Campochiaro, 1987). To date, receptor-coupled activation of phospholipase C for acetylcholine or, indeed, for any other agonist, has not been demonstrated in RPE cells of any species, although the recent report by Friedman, Hackett and Campochiaro (1988) showed that acetylcholine and carbachol cause an increase in intracellular calcium concentrations of cultured RPE cells, thus suggesting the occurrence of such receptors. The present study provides proof of the presence of mu~a~nic receptors coupled to &heactivation of phospholipase C in human RPE cells.
1119
N.N.
1120 MATERIALS
OSBORNE
AND METHODS
Methods Preparation of primary cultures of human RPE. Post-morten adult eyes (received l-2 days
following death) were stored overnight at 4°C and the transferred to a sterile cabinet. The cornea just below the ora serrata was dissected and the lens, vitreous and retina carefully removed. The eye was carefully rinsed with about 500 ~1 PBSA solution (137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH,P0,*H20, 7 mM Na,HPO,, pH 8.2). A 0.25% trypsin solution (in PBSA) was then added to the eye cup (to below the cut edges to avoid contamination) and incubated for 60 min at 37°C in a moist 5% CO2 atmosphere. Most of the trypsin solution was carefully removed and replaced with a culture solution (120 ml distilled water, 20 ml foetal calf serum-heat inactivated, 10 ml 285 mM sodium bicarbonate solution, 20 ml 10 x Hams FlO solution, 2 ml 200 mM glutamine, 6 ml 0.44 M glucose, 2 ml of 10 mg/ml gentamycin, pH 7.4). With a brush the RPE cells were suspended in the culture solution, transferred to a tube and centrifuged at 70g for 7 min at 4°C. The pellet was resuspended in a fresh culture solution and cells were grown in 6 ml Hams FlO medium in 25 cm* tissue culture flasks (from Sterilin) to passage four. Cells (from passages 2-4) were grown on 13 mm diameter glass cover-slips [previously coated with a solution (10 ,ug/ml) of poly l+lysine] in Multidish (Falcon 3008) wells at a density of 5-10 x lo4 cells per coverslip. The cultures were grown at 35.5”C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was changed at 3-day intervals. Stimulation of inositol phosphates
(InsPs).
Coverslips containing confluent cultures (4-6 days subsequent to plating) were incubated for 15 hr at 35.5”C (in a 5% CO? humidified atmosphere) in Eagles Minimal Essential medium (MEM) plus glucose (final concentration 33 mM) containing 0.1 PM ‘H-myoinositol. An incubation time of 15 hr was routinely used since trial experiments showed that at this time isotopic equilibrium was maximal. The cultures were then rinsed with MEM solution containing 5 mM LiCl (lithium/MEM solution) and then preincubated in the same solution for 15 min. Each culture was then transferred to another Falcon multiwell which contained 290 ~1 lithium/MEM solution plus a further 10 ~1 of this solution either alone or containing an
et al.
antagonist. The cultures were then incubated for 10min at 35.5”C, after which a further 10~1 lithium/MEM solution containing agonist, was added. Control cultures were simply supplied with vehicle solution. Each coverslip containing a culture was therefore incubated with a total volume of 310 ~1 of solution. After an incubation period of 45 min at 35.5”C the reaction was stopped by transferring cultures on two or three coverslips into 1 ml chloroform methanol (1: 2, v/v) present in a glass tube. Subsequently, the media from two or three wells plus an equal volume of chloroform were added to each glass tube. The coverslips and RPE cells were crushed with a glass rod inside the glass tubes, vortexed, and then left at 5°C overnight to allow the complete separation of the aqueous phase from the lower liquid phase. The water soluble 3H-InsPs were then extracted using Dowex as described by Berridge, Downes and Hanley (1982). Materials
Myo-(2-3H)-inositol, 12 Ci/mmol was from Amersham International, U.K. Foetal calf serum, 10 x Hams FlO, MEM and glutamine was brought from GIBCO, U.K. Gentamycin, pertussis toxin and phorbol esters were from Sigma, U.K. All other chemicals were purchased from Sigma or from BDH (U.K.). RESULTS
The identity of the human RPE cultures from a 4th passage 6 days after being plated and “stained” immunohistochemically for the localization of vimentin-immunoreactivity is shown in Fig. 1 (see Osborne, Morgan, Ghazi & Beaton, 1988b for details of methodology and source of antibody). The cells appear homogenous in nature and are confluent. (a) E#ect of carbachol on the accumulation of ‘H-InsPs
Addition of carbachol to the RPE culture induced a dose-dependent increase in ‘H-InsPs accumulation in the presence of 5 mM LiCl (Fig. 2). Carbachol at a concentration as low as 10 fl M evoked a 110% increase in InsPs accumulation within 45 min of incubation, and, at the saturation concentration (1 mM), the increase was 590% relative to basal level. From the dose-response relationship, the ECSo value of carbachol was found to be about 70 p M.
Fig. I. Immunohistochemical localization of vimentin-like immunoreactivity in a 20 day-old human pigment epithelium culture. It can be seen that the cells are confluent at this stage in the culture. Scale bar 50pM.
1121
Phosphoinositide Dose
response
curve
turnover in human RPE cells
1123
RPE
for
5 ii
500 250
E’
i Corbachol concentration
(@A)
Fig. 2. Concentration-response relationship of carbacholinduced 3H-InsPs accumulation in pigment epithelium cells. The cells were labelled overnight for 15 hr with 3H-myoinositol and then washed to remove the isotope. After preincubation with 5 mM LiCl at 355°C for 15 min, various concentrations of carbachol were added and the cells were further incubated for 45 min. The accumulation of ‘H-InsPs was then measured using Dowex as described by Berridge et al. (1982). The data presented are mean + SEM of three independent experiments which were performed in triplicate. The basal activity in the absence of carbachol was expressed as 100% and was 3 15 k 100 dpm per sample.
The carbachol-induced InsPs accumulation was linearly increased for the first 45 min of incubation and continued to increase in a non-linear fashion up to 120 min (Fig. 3); at the latter time, a 610% stimulation was observed. Separation of ‘H-Ins- l-P, 3H-Ins-2-P and 3H-Ins-3-P following the method of Berridge et al. (1982) showed that most of the radioactivity was associated with ‘H-Ins- 1-P after 45 min stimulation by carbachol (see Fig. 4). This was also the case after 15 min stimulation with carbachol, whereas after a period of only 5 min the radioactivity was not so unequally distributed between the different 3H-InsPs. Effect 700
of
IOOpM
corbachol
r-
5 mill
15min
45 mln
Fig. 4. Comparative distribution of 3H-Ins-l-P, ‘H-Ins-2-P and ‘H-Ins-3-P following stimulation of pigment epithelium cells for 5, 15 and 45 min with 100 PM carbachol. The methods used were as described in the text and Fig. 1, except that the accumulated )H-InsPs was eluted from the Dowex column by stepwise elution, using different concentrations of ammonium formate and formic acid as described by Berridge et al. (1982). Results are mean + SEM of three independent experiments.
Atropine, a nonselective muscarinic antagonist and pirenzepine, a putative M, muscarinic receptor antagonist were assessed for their ability to block the increased accumulation of InsPs evoked by carbachol. Table 1 shows that while both atropine and pirenzepine do antagonize the effect of carbachol, atropine is the most effective. Prazosin, the a,-adrenergic receptor antagonist and ketanserin, the 5-HT, receptor blocker failed to influence the carbacholinduced response at concentrations as great as 10pM. (b) Desensitization of carbachol-mediated phosphoinositide turnover
In order to assess the desensitization of carbachol-induced accumulation of InsPs, cultures were pretreated with 100 PM carbachol throughout the period of incubation with radioactive inositol. Thereafter the cells were washed
T
Table 1. Effect of various antagonists on the carbachol-induced stimulation of ‘HInsPs accumulation in pigment epithelium cells. The concentration of carbachol was 1OOpM. Results are expressed in terms of percentage inhibition and are the means of three separate experiments each carried out in triplicate
I 0
I
I
15
30
1 45
1
1
I
I
’
60
75
90
105
120
Time (mh)
Fig. 3. Time course of carbachol-induced 3H-InsPs accumulation in pigment epithelium cells. Experimental conditions were described in the text, except that cells were incubated for various periods of time in the presence of 100 PM carbachol. The data presented are mean + SEM of a triplicate experiment, repeated three times.
Antagonist Atropine (1 PM) Atropine (5 FM) Pirenzepine (5 p M) Pirenxepine (10 PM) Ketanserin (10 PM) Prazosin (10 bM)
% Inhibition of carbachol response 62 k 9 78 + 7 31+9 48 k 7 10 f 4 8z%4
N.B. Ketanserin and prazosin effects were not significant.
1124
N.N. OSBORNEet al.
laJ n
n-
2000
[]
Basal
"6
1750
•
Stimulated
._~
1500
H ~
1250
~ ~ 400
1000 ~,~
700
~
~
300
5 20o
500
N ~
o
Control
P re-- exposure to CCh
[ ] I/~M ÷ Stourosporine [ ] d/zg/ml
500
750
250
[ ] Control I&-~OA/~M • I/~M
100
1iiniiD1i PMA
Inactive Phorbol Ester
PTX
Fig. 5. Carbachol-induced 3H-InsPs accumulation in retinal pigment epithelium cells pre-exposed to muscarinic receptor agonist carbachol. Cells were incubated in the absence or presence of carbachol (100#M) for 15hr during the cell labelling and then washed three times before challenging them with 100 p M carbachol (stimulated values). It can be seen that while the basal level of 3H-InsPs was not influenced by pre-exposure to carbachol, the stimulated value was much reduced. Results are mean ___SEM of three independent experiments.
Fig. 6. Effect of phorbol esters and pertussis toxin on carbachol-induced stimulation of 3H-InsPs accumulation. Labelled cells were exposed to phorbol esters 15 min before being challenged by the addition of 100 #M carbachol. In the experiments with staurosporine, the substance was added 5 min before the addition of phorbol esters. In the case of the pertussis toxin (PTX) experiment, the substance was added to the culture during the 15 hr incubation with 3H-myoinositol. Results are mean values __+SEM for 3~J, separate experiments.
free o f c a r b a c h o l a n d r a d i o a c t i v i t y a n d then challenged with c a r b a c h o l in the n o r m a l w a y in the presence o f LiC1. A s s h o w n in Fig. 5, p r e - e x p o s u r e to c a r b a c h o l causes a m a r k e d i n h i b i t i o n o f 3H-InsPs a c c u m u l a t i o n i n d u c e d b y s u b s e q u e n t s t i m u l a t i o n with 100 # M c a r b a c h o l . Basal values for 3H-InsPs were n o t d r a s t i c a l l y affected b y p r e - e x p o s u r e to c a r b a c h o l . T h e results t h e r e f o r e suggest t h a t p r e - e x p o s u r e to c a r b a c h o l causes a d e s e n s i t i z a t i o n o f the receptors.
are m i l d e r a n d p i l o c a r p i n e is the least effective. T h e influence o f s e r o t o n i n a n d q u i s q u a l a t e was negligible w h e r e a s n o r a d r e n a l i n e s t i m u l a t e d 3HInsPs, b u t m u c h less effectively t h a n c a r b a c h o l .
(c) Comparison of the effect of carbaehol and other agonists on phosphoinositide turnover T a b l e 2 shows the c o m p a r a t i v e effects o f 100 # M o f v a r i o u s cholinergic agonists ( c a r b a chol, p i l o c a r p i n e , m u s c a r i n e a n d arecoline), n o r a d r e n a l i n e , s e r o t o n i n a n d q u i s q u a l a t e on the s t i m u l a t i o n o f 3H-InsPs a c c u m u l a t i o n in R P E cultures. T h e m o s t effective substance is c a r b a chol. A l t h o u g h the o t h e r cholinergic substances s t i m u l a t e 3H-InsPs a c c u m u l a t i o n , their effects Table 2. Effects of various agonists on 3H-InsPs accumulation in pigment epithelium cells. Results are expressed in terms of percentage accumulation above basal levels. Results are mean values __+SEM where n = 4 Substance (I00 pM) Carbachol Pilocarpine Muscarine Arecoline Noradrenaline Serotonin Quisqualate
% Stimulation of 3H-InsPs 408 _+32 94 __+28 226 + 55 154 + 39 84 _+22 29 4- 18 33 _+ 19
(d) Effect of phorbol esters and pertussis toxin T h e p r o t e i n kinase C a c t i v a t o r , P M A (4//p h o r b o l 12-myrisate 13-acetate) a n d the biologically inactive p h o r b o l ester, 4 ~ - p h o r b o l 12,13 d i d e c o n o a t e were e x a m i n e d for their effects on the c a r b a c h o l - i n d u c e d s t i m u l a t i o n o f 3H-InsPs a c c u m u l a t i o n . P M A at c o n c e n t r a t i o n s o f 0.1 a n d 1/~M i n h i b i t e d 3H-InsPs a c c u m u l a t i o n b y 35% a n d 4 6 % respectively w h e r e a s the inactive p h o r b o l ester failed to a t t e n u a t e the response in the s a m e c o n c e n t r a t i o n r a n g e (Fig. 6). A t a c o n c e n t r a t i o n o f 1 # M s t a u r o sporine, a p r o t e i n kinase C i n h i b i t o r (see H i d a k a & H a g i w a r a 1987; H u a n g 1989), nullified the effect p r o d u c e d by P M A . Pertussis toxin is k n o w n to i n a c t i v a t e Gi p r o t e i n t h r o u g h A D P r i b o s y l a t i o n o f the alphai s u b u n i t ( K a t a d a & U i 1982). The a d d i t i o n o f pertussis toxin (1 # g per ml) to the r a d i o a c t i v e inositol over a p e r i o d o f 15 hr did n o t affect the extent o f 3H-InsPs a c c u m u l a t i o n i n d u c e d b y c a r b a c h o l : basal values were n o t significantly affected by pertussis toxin either. DISCUSSION These results p r o v i d e the first p r o o f o f the presence in c u l t u r e d h u m a n R P E cells o f m u s c a r i n i c r e c e p t o r s linked to p h o s p h o i n o s i t i d e turnover. T h e ECs0 value o f c a r b a c h o l for
Phosphoinositide
turnover in human RPE cells
InsPs accumulation is lithium-dependent about 70pM and maximum stimulation for 100 PM carbachol reached 6-fold after about lOOmin. This agonist potency and the degree of stimulation are similar to what is found in other systems, including retinal slices (Osborne, 1988; Cutcliffe & Osborne, 1987), brain slices (Batty & Nahorski, 1985), astrocytes (Pearce, Cambray-Deakin, Marrow, Grimble & Murphy, 1985) and NCB-20 cells (Chuang, 1986). The carbachol-induced response was inhibited by two muscarinic antagonists, atropine and pirenzepine, the latter being weaker. This suggests that the carbachol-induced phosphoinositide turn-over might be mediated in human RPE cells through Ml-type muscarinic receptors. This would be consistent with the observation made by Friedman et al. (1988) that stimulation of muscarinic receptors in human RPE leads to a mobilization of internal calcium levels, an observation most often associated with pharmacologically characterized M,-type receptors. In addition, receptors mediating an increased accumulation of InsPs would be expected to result in a simultaneous increase in the mobilization of internal calcium levels caused specifically by the action of Ins-3-P (see Berridge & Irvine, 1989; Osborne & Ghazi, 1990). Of the muscarinic agonists tested, carbachol was the most effective in stimulating InsPs accumulation in human RPE cultures. The order of effectiveness for the various muscarinic agents is similar to what has been described for the stimulation of InsPs in retinal slices (Osborne, 1988) and may be explained by the fact that the agents used range from being partial agonists (e.g. pilocarpine) to full agonists (e.g. carbachol). The specificity of the cholinergic receptors is exemplified by the finding that other antagonists of receptors linked to stimulation of phosphoinositide turnover, viz. ketanserin (5-HT, receptor antagonist) and prazosin (a,-receptor agonist) were ineffective in blocking the carbachol effect at concentrations as great as 10 v M. In comparison with carbachol, other potential agonists such as noradrenaline (a,-receptors), serotonin (5-HTz receptors) and quisqualate (quisqualate receptors) hardly stimulated phosphoinositide turnover (see Table 2). This differs from what occurs in the retina, where the order of effectiveness in stimulating InsPs is quisqualate > carbachol > noradrenaline > serotonin (see Osborne & Ghazi, 1990; Osborne, 1990; Ghazi & Osborne,
1125
1988). The present results therefore show that @,-type, 5-HT,-type and quisqualate-type receptors linked to InsPs production are either absent or present in low amounts in human RPE cells. Further experiments are necessary to clarify this point. The carbachol-induced response in the RPE cells could be desensitised by pretreatment with carbachol. This is not a surprising finding, although it is not known by which mechanism this occurs. It is conceivable that the continuous accumulation of diacylglycerol and InsPs results in a persistent activation of muscarinic receptors, which leads to an activation of protein kinase C and phosphorylation of specific proteins. The protein phosphorylation may trigger down-regulation of muscarinic receptor binding sites, or alternatively, an internalization of receptors which could be associated with the normal recycling process of receptors. The biologically active phorbol ester, PMA, was able to attenuate carbachol-induced InsPs accumulation, whereas the biologically inactive phorbol ester, 4a -phorbol 12,13-didecoate, was ineffective in causing the effect in RPE cells. The PMA effect was nullified by the PKC inhibitor, staurosporine. These data are analogous with results obtained by various investigators using other receptor systems (Ghazi & Osborne, 1989; Roth, Nakaki, Chuang & Costa, 1986; Chuang 1986; Labarca, Janowsky, Pate1 & Paul, 1984). It is well established that active phorbol esters act on protein kinase C to induce their effects (Nishizuka, 1984). The inhibitory effect produced by PMA shows that protein kinase C activation alters muscarinic receptor-mediated responses in human RPE. Likely sites of action of activated protein kinase C include: (a) the receptor, (b) GTP binding protein, (c) phospholipase C or (d) indirectly through, e.g. c-AMP 2nd messenger systems. Further experiments are clearly necessary to discover whether any of these mechanisms are involved. Irrespective of the process, the present results suggest that diacylglycerol formation during physiological muscarinic-induced activation in RPE cells may have a negative feedback on the production of InsPs via action of protein kinase C. Such a process would help maintain the cellular physiological homeostasis. As in other systems (Schimmel & Elliott, 1986; Uhing, Prpic, Jiang & Exton, 1986), pretreatment with pertussis toxin for as long as 15 hr did not affect basal or carbacholinduced InsPs accumulation in RPE cells.
N. N. OSBORNE et al.
1126
Under similar conditions, the carbacholinduced attenuation of adenylate cyclase activity was reduced by more than 50% in iris/ ciliary muscle (Tobin & Osborne, 1988). The lack of effect of pertussis toxin in the muscarinic receptor-mediated InsPs accumulation in RPE cells is in contrast to the inhibition by pertussis toxin of receptor phosphoinositide turnover in some other systems (Brass, Laposta, Banga & Rittenhouse, 1986; Pfeilschifter & Bauer, 1986) and suggests that the GTP regulating protein of the MI-type muscarinic receptors in RPE cells may be distinct from the Gi protein. In conclusion, the present results show that muscarinic receptors linked to InsPs accumulation exist in human RPE cells. It remains to be determined exactly where these receptors are local&d on the RPE cells in order to determine their physiological relevance. Should the receptors be exclusively associated with the apical surfaces of the RPE cells then consideration has to be given to the possibility that the photoreceptors provide the source of the natural agonist (viz. acetylcholine). Present evidence suggests, however, that photoreceptors of mammals are not choline@, though it is known that in lower vertebrates, such as mudpuppy and turtle, certain photoreceptors may synthesise acetylcholine (see Pure, 1985). Acknowledgements-We
are extremely grateful to the British Retinitis Pigmentosa Society and the Royal National Institute for the Blind for financial support.
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Nishizuka, Y. (1984). The role of protein kinase C in cell surface receptor signal transduction and tumour promotion. Nature, London, 308, 693698. Osborne, N. N. (1988). Muscarinic stimulation of inositol phosphate formation in rat retina: Developmental changes. Vision Research, 8, 875-888. Osborne, N. N. (1990). Stimulatory and inhibitory actions of excitatory amino acids on inositol phospholipid metabolism in rabbit retina: Evidence for specific quisqualate
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