Exp. Eye Res. (1975) 20,63-72
The Effect of Retinol and Acetylsalicylic Acid on the Release of Lysosomal Enzymes from Rat Retina in vitro A. J; DEWAR, GILLIAN
BARRON AND H. W. READING
MRC Brain Metabolism Unit, Department of Pharmacology, University of Edinburgll, 1 GeorgeSquaw, Edinburgh, EH8 9JZ, U.K. (Received18 September1974, London) Retinol caused an increased release of fi-glucuronidase, ,&galartosidase and hexosaminidase from rat retinal lysosomes. Maximal labilization was produced by 1.5 rg/mg wet weight of retinal tissue. Acetylsalicylic acid in concentrations of 0*25-0.50 mM produced a significant stabilizing effect on rat retinal lysosomes both in the absence and presence of 1.5 pg/mg of retinol. It is suggested that acetylsalicylic acid and other anti-inflammatory drugs may merit further investigation as possible agents for combating retinal degeneration.
1. Introduction Degeneration of the retina in the strain of albino dystrophic rats known asCampbells (Bourne, Campbell and Tansley, 1938), is associatedwith an increase in the level of “free” lytic enzymeswhich appear to originate from lysosomesin the adjacent pigment epithelium (Burden, Yates, Reading, Bitensky and Chayen, 1971). It has been suggested (Reading, 1970) that the degeneration of the visual cells is produced by breakdown of lysosomal membranesin pigment epithelium and retina. This breakdown is thought to be due to an abnormal build-up of vitamin A (retinol) in the pigment epithelium which arisesfrom the action of light on an unusually labile type of visual pigment (Reading, 1966). It has been demonstrated that depriving Campbell rats of light, significantly retards the progress of the retinal degeneration (Dowling and Sidman, 1962; Dewar and Reading, 1974a). The presence of pigment in the eye in rats of a pigmented dystrophic strain known as Hunters also retards the progress of the degeneration (Yates, Dewar, Wilson, Winterburn and Reading, 1974). It has been demonstrated that retinol labilizes lysosomesin a variety of tissues (Fell, Dingle and Webb, 1962) and recently Vento and Cacioppo (1973) described the increasedreleaseof lysosomalenzyme produced by retinol in bovine retina and pigment epithehum in vitro. There is evidence that anti-inflammatory agents have a stabilizing effect on lysosomesand can retard the labilizing effects of histamine in inflammation (Chayen, Bitensky and Ubhi, 1972). In addition, a clinical study by Powell and Field (1964) revealed that a group of diabetic patients suffering from rheumatoid arthritis and requiring continuing large dosesof salicylates had a reduced incidence of retinopathy compared with the general diabetic population. This paper presents the results of a study of the effects of retinol and acetylsalicylic acid on rat retinal lysosomes. Part of this work has been presented as a preliminary communication (Dewar and Reading, 1974b). 2. Materials and Methods The e$ect of retinol on retinal
lysosomal stability
Sighted albino Wistar or Piebald Virol Glaxo (PVG) rats of either sex and of an age range I-3 months were used throughout. from both strains of rat.
Similar 63
results were obtained
with the retinas
6-l
A. J. DEIVAII,
c:. AAI’,KOS ASI) Il. IT. REAlJIx’C;
The rats were killed by tlecapitation. Retinas were removed aud homogenixetl iI1 12 url ice cold 0.25 &I-sucrose/g wet weight retina tissue for (1.5 min at ~OOO rev/nliu. The prc~cipitates were discarded and the superuatant centrifuged for two further .‘)-nlitl period% at IICUOg. The supernntant was t,heu split into 0.5 ml irliquots (eac:h containing the equivaletit of 46 nig of original tissue) arid these were added to tubes couta,inillg 293 ml (I.d5 M-sucrose plus kither O.l(y” V/V Triton Y-lCJ0 or O-2.5 pg/mg origiual tissue retinal. The retinol was dissolved in the minimutu volume of ethauol. This volume of ethanol was al+o added to the tubes not containing retinol. In most experiments all tubes were incubwtetl at 37°C for 45 min under a continuous flow of nitrogen but in some, the incubation time was varied from O--l80 min. After incubntion, each tube was cooled t.o (1°C ant1 t,he contents then centrifuged at 15 000 g for 20 min to pellet the intact lysosomes. To nsse”& the damage to the incubated lysoaomes the activities of four lysoeomnl enzymes: P-glucuronidase, acid phosphntase, ,&galactosidase and hexosaminidase mere measured. All activities were expressed as a percentage of the total releasable activity, i.e. as the percentage of the activity in the supernutaut derived from the tubes treated with 0.1 % Triton S-100.
/3-G’luczcro~l.idnse. /SGlucuronidase activity was measured by incubating 1 ml of each lysosomal-free supernatant with 0.2 ml 0.1 ;\I-phenolphthalein-mono-p-glucuronate (Sigma) in 1.6 ml 0.1 M-sodium acetate buffer, pH 4.5 for 19 hr at 37°C. Prior experiments had confirmed that the reaction proceeded in a linear manner over this period. At the end of the incubation protein was precipitated by the addition of 1 ml Sgb trichloracetic acid. Two ml 0.4 M-glycine buffer (pH 10.45) were added to 2 ml of the protein-free supernatant. After 30 min the extinction at 540 nm was determined. This method was derived from that, of Vento sud Cacioppo (1973). Acid phospl~atase. Acid phosphatase was supernatnnt to 1 ml citrate buffer, pH 5.5 which had been preincubated for 10 min at and 45 min incubation at 37°C by addition measured spectrophotometrically at 400 nm.
assayed by adding l-ml aliquots of diluted containing 0.1 M-paranitrophenyl phosphate 37°C. The reaction was stopped after 15, 30 of 1 ml 0.5 RI-NaOH. The p-nitrophenol was
/SGulactosidase. p-Galactosidase was measured by adding 0.5 ml of supernatant to 1 ml of 0.1 &I-glycine-HCl buffer pH3 containing 1 x 10~~ af-4-methylun~belliferylgalactosicle (MUG) plus 0.5 ml distilled water which had been preincubated at 37°C for 10 min. After incubation at 37°C for 60 min, 2 ml 0.1 H-Na,CO, was added and the fluorescence read at an excitation wavelength of 365 nm and an emission wavelength of 450 nm on a PerkinElmer spectrophotofluorimeter. This method is an adaptation of that of Yates (1973, Personal Communication) for measuring /%galactosidase in rat brain. This, in turn, was a modification of the method of Robins, Fischer and Lowe (1961). Hexosaminidase. Hexosaminidase was measured by adding 0.2 ml of the supernatant to 0.8 ml of 2.5 mM-4-methylumbe~iferyl-2-acetamido-2-dioxy-~-D-glucopyranoside in a buffer composed of 50 parts 0.2 M-Na,HPO, and 50 parts 0.1 M-citric acid. After 30 min incubation at 37°C the reaction was stopped by the addition of 3 ml 0.1 M-Na,CO,. The fluorescence was read at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. The effect of acetylsalicylic
acid on retinal lysosome
stability
The stability of lysosomes from normal retinas was examined in the presence of O-10 mMacetylsalicylic acid and in the presence of O-1 mM-aCetyhdiCyh acid plus 1.5 pg/mg original tissue retinol. The methods of preparation, incubation and enzyme assay were identical to those described above.
RELEASE
OF
LYSOSOMAL
ENZYMES
FROM
RAT
RETINA
65
The effect of retinol and acetylsalicylic acid on the activity of released lysosomal enzymes Retinal lysosomes were prepared and incubated for 45 min at 3’7% with 0.1% v/v Triton X-100 to release the lysosomal enzymes. After centrifuging the cooled incubation mixture at 15 000 g for 20 min, the supernatant was removed and split into three portions. One portion was used to test the effect of a range of acetylsahcylic acid concentrations on the activities of fi-glucuronidase, /Lgalactosidase, hexosaminidase and acid phosphatase, a second was used to test the effect of a range of retinol concentrations, and the third portion was used to determine the effect of a combination of acetylsalicylic acid and retinol.
3. Results The effect of retimol on lysosomal stability Retinol significantly increased the release of /3-glucuronidase, hexosaminidase and p-galactosidase from retinal lysosomes (Fig. 1). An increased release of /Sglucuronidase was observed with all concentrations of retinol tested but in the case of pgalactosidase and hexosaminidase a significantly increased release was observed only with retinol concentrations of 1.0 pg/mg and above. In all three case maximal release was obtained with 1.5 pg/mg retinol in the incubation medium. 8
“0 8
0
05
IO Concentration
I.5 of retinal
2.0 pg/mg
2.5
wet weight
FIG. 1. The effect. of retinol on the release of enzymes from retinal lysosomes during a 45-min incubation at 37”~. Points represent mean + S.D. of the number of determinations shown in brackets. The levels of significance (t test) of the increased release produced by 15 pg/mg were: fl-glucuronidase t = 7.66. P ( O@Ol; hexosaminidase t = 4.35, P < 0.01: j3-galactosidase t = 6.42, P < 0.001. The increase in acid phosphatase release was not significant. ,%Glucuronidase, 0 - - - 0 ; acid phosphatase, x -x : p-galactosidase, 0 -0 ; hexosaminidase, l __ 0.
The retinol failed to have a significant effect on the releaseof acid phosphatasesince. even in the absence of retinal, the bulk of the lysosomal acid phosphatase had entered the supernatant fraction during the course of the 45-min incubation. It was possible that the increased lysosomal enzyme activity in the supernatant fraction after retinol treatment was not a releasephenomenonbut an activation of the releasedenzymes. However, the results presented in Fig. 2 suggestthat this is unlikely, Retinol appeared to have very little effect on the activities of the releasedenzymes E
66
A.
.c >r .5 z D
J.
DEW’AR,
G. BARROS
AED
H.
\2’. READIN(:
90 eo-
8 :: 3
70-
i$
50-
.2 .g
40-
=i
30
GO-
I 0.5
0
I I.0
Concentration
2. The effect of retinol duplicates. Acid phosphatase, glucuronidase, l - - - 0. FIG.
I 15
of retinol
pg/mg
on the activity of released x x ; hexosaminidase,
TABLE
I 20 original
retinal
l
2.5
tissue
lysosomes. Points represent __ l ; /3-galactosidase, 0 --
mean of 0 : ,%
I
The effect of acetylsalicylic acid on /%glucuronidasereleasefrom retilzal lysosomesduring a 45-m& incubation at 37°C Concentration ASA (mM) 0 (Control) 0.05 0.10 0.25 0.50 1.00 2.50 5.00 7.50 10.00
*P
< 0.01.
of
o!oRelease in absence of retinol 49.5 54.1 65.0 17.1 21.5 34.9 24.1 71.3 79.6 123.0
t P < 0.025. Values represent
* + i * & + & (2) (2) k
22 (14) 2.6 (3) 1.4 (3) 5.4 (5)* 11.5 (6)t 13.9 (3) 6.1 (3):
4.9 (3)
%Release with 1.5 pg/mg retinal in incubation medium 112.1 104.2 119.3 54.3 26.6 88.5 72.6 70.0 70.0 121.6
$ P < 0.05. § P < 0~0001. mean * S.U. n shown in brackets.
* * * * += & (2) (2) (2) f
94 33.2 14.7 9.2 15.9 8+3
(9) (3) (4) (4)s (6)s (4)**
1.08 (4) ** P < 0.005.
and certainly not enough to explain the results shown in Fig. 1. However, the fact that the release of P-glucuronidase produced by 1.5 pg/mg retinol slightly exceeds the theoretical 100% (Fig. 1) doessuggestthat retinol may slightly activate the enzyme as well as promoting its release.This is not borne out by the data in Fig. 2 but the possibility that the presence of Triton X-100 may abolish any activating effect of retinol cannot be excluded. The effect of acetylsalicylic acid on lysosomestability Acetylsalicylic acid at concentrations of 0.25-0.50 mM significantly reduced the releaseof all the lysosomal enzymes tested (Tables I and II). At concentrations less
86.3 87.3 82.6 47.9 61.0 93.5
0 0.05 0.10 0.25 0950 1.00
of retinol
f 15.1 (11) * 10.3 (4) * 27.7 (5) * 14.0 (ll)* * 16 (5) + 15.5 (4)
In absence
u/ORelease
Cm@
c011c. ASA
of acid
The e#ect of ncetylsalicylic
II
96.2 77.5 63.5 43.1 45.7 53.0
Values
-J= (2) (2) + & +
16.1
(5)
retinol
* P < 0.001. represent
6.6 (5)t 4.9 (5)$ 21.1 (3)
+ I.5 pg/mg
phosphatase
mean
& & & * + -+ 10.8 5.7 7.1 8.9 13.8 7.5
(8) (3) (4) (?I)* (S)$ (3)
or retinol
t P < 0.005. k S.D. where
41.3 46.3 41.7 11.8 22.4 50.6
In absence
~/oRelease
$ P < 0.05. appropriate.
78.4 53.0 44.0 13.1 23.2 45.3
& * f f + &
(5) (3)$ (3)s (4)* (4)* (3)*
retinol
ti shown
in brackets.
41.6 34.3 30.3 0.37 9.2 15.1
In absence
retinal
i & f + & *
5.4 (4) 4.7 (3) 1.9 (3)$ 0.44 (3)* 0.94 (3)* 0.47 (3)*
of retinol
%Release
release from
5 P < 0.025.
5.6 14.1 15.5 6.4 2.2 1.8
+ 1.5 pg/mg
of 8.gatactosidasc
n&d on rtcid phosphatase /3-galaetosidase and hexosamirkdase a 45min. incubation at 37°C
TABLE
71.6 68.0 73.6 19.0 19.0 39.3
* & 3 & & &
+ 1.5 pg/mg
of hexosaminidase
5.3 1.4 4.4 12.7 2.5 2.6
(3) (3) (3)s (3)s (3)t (3)s
retinol
lysosomes during
0 (Control) 0.05 0.10 0.25 0.50 1.00 5.00 10.00
_-__
Concentration acetylsalicylic acid (mu)
of
100 92 98 92 96 95 94 92
In absence retinol
___-
of +l+
100 97 92 85
92
f&w retinol
Acid
ret&al
108 100 102 103 92
the mean
Values
+ 1.5 pgbg retinol
of duplicates.
100 104 109 109 109 114 104 104
Activity
of
95 100 109 100 -
In absence retinol
100 106 109 106 100 102 106 106
of cm&-ok
of
102 98 96 -
99
retinal
-: I.5 pg/mg
Hexosaminidase
expressedas percentage
+ 1.5 pglmg retinol
,%Galactosidase
enzymes.
In absence retinol
lysosomal
111
100 102 104 105 104 101 99 90
represent,
of
phoaphatase
of released
In absence retinol
acid on the activity
p-Glucuronidase
The e$ect of ucetylsalicylic
TABLE
RELEASE
OF
LYSOSOMAL
ENZYMES
FROM
RAT
RETINA
69
than 0.10 mM it had no effect except on hexosaminidase release. The release of /3glucuronidase was reduced by acetylsalicylic acid concentrations as high as 2.5 mM hut at higher concentrations it appeared to have a labilizing effect. Acetylsalicylic acid significantly stabilized retinal lysosomes against the labilizing action of 1.5 pg/mg retinol. In the absence of acetylsalicylic acid retinol produced an enzyme release ranging from 78 to ll2o/o but in the presence of the optimum concentration of acetylsalicylic acid this was reduced to between 13.1 and 43.1%. In the case of /3-glucuronidase release 0.25-7.50 mM had a stabilizing effect but the maximal effect was produced by 0.50 mM. At concentrations in excess of 7.5 mM there was no stabilizing effect. In the case of the other lysosomal enzymes, 0.25 mM-acetylsalicylic acid appeared to be the most effective stabilizing concentration. Acetylsalicylic acid had little effect on the activity of lysosomal enzymes after their release (Table III) thus indicating that its effect is to stabilize retinal lysosomes rather than inhibit the activity of the enzymes after release.
-_ _0 Preparation and centrifugation
30
45
120
60 Duration
of incubation
180
(mid
FIG. 3. The effect of incubation time on the effects of Triton X-100, retinol and acetylsalicylic acid on retinal lysosomal enzyme release. Points represent mean (f S.D. where applicable) of the number of determinations shown in brackets. (a) Hexosaminidase, Triton X-100, 0 l ; 1.5 pg/mg retinal, m---m; noadditions, A.... .A; 1.5 pg/mg retinol + 0.26 mu-acetylaalicylic acid, n---A ; 0.25 mM-acetylsalicylic acid, 0 -0. (b) p-Galactosidase. Triton X-100, 0 __ 0 ; 1.5 pg/mg retinal, m---w;noadditions,A.... . A; 0.25 m&x-acetylsalicylic acid. 0 __ 0 ; 0.25 mM-acetylsalicylic acid, A-/J.
io
A. J.
DEWAR,
Q.
BARROK
AND
H.
W.
READISC:
The e$ect of imxbation time on, the effect of Tritocn-X 100, retinol and acetylsalicylic acid lysosome stability This wasinvestigated usingP-galactosidaseand hexosaminidaseas enzyme markers. The results are shown in Fig. 3. Triton X-100 (0.1%) causedmaximum releaseof these enzymes within 45 min. During incubations exceeding 1 hr in duration, the Triton X-100 releasedactivity fell, presumably due to a direct inhibitory effect of Triton X-100 on the enzyme activity after release.A similar drop in activity was seenin the incubations with 1.5 pg/mg retinol. The lysosomal stabilizing effect of 0.25 mMacetylsalicylic (both in the presence and absenceof 1.5 pglmg retinol) apppeared to be maintained during a 3 hr incubation at 37°C. There are marked differences in the enzyme activity releasedin the presenceof Triton X-100, retinol and acetylsalicylic acid even in the absenceof an incubation at 37°C. This indicates that these agents affect lysosomestability during the centrifugation at 15 000 g. on retinal
4. Discussion We have demonstrated that retinol has a profound labilizing effect on rat retinal lysosomes.This is in agreement with the findings of Vento and Cacioppo (1973) who found that 1.5 pg/mg retinol produced the maximum labilizing effect on bovine retinal lysosomes.However, Vento and Cacioppo considered that retinol had a considerable activating effect on /3-glucuronidasesince the releasedactivity of this enzyme was as much as 28Oo/oof the activity releasedby sonication. The hypothesis outlined in the introduction suggeststhat the elevated retinol levels in the pigment epithelium of the dpstrophic rat results in an increased release of lysosomal enzymes. The present results demonstrate that retinol does labilize lysosomesbut doessoin concentrations considerably larger than the retinol levels reported by Reading (1966, 1970) who found that the tissue retinol level of the pigmented layers of 2-week-old dystrophic rats was ll-lSpg/g wet weight and in normal sighted rats approximately 7 lug/g wet weight. However, these absolute in vivo levels are almost certainly an underestimate. The pigment layers used in the assay consisted of pigment epithelium, choroid and sclera (i.e. the back of the eye minus the retina) and consequently the figures obtained must have been subject to oonsiderable dilution due to the presenceof excesstissue. In addition the pigment layers sample did not include the extralamellar material from the dystrophic rats. Thus at present it is impossible to say with certainty whether the retinol effect we observe unequivocally in vitro alsooccurs in vivo in the dystrophic retina. However, it is probable that the retina and the extralamellar material of the dystrophic rats contain far higher concentrations of retinol than the pigment epithelium and that local concentrations of retinol in the retina are sufficient to labilize lysosomes. Our results indicate that acetylsalicylic acid significantly stabilizes retinal lysosomes at certain concentrations but labilizes them at higher concentrations. The optimum stabilizing concentrations found in our experiments was the same as that reported by Tanaka and Lizuka (1968) to stabilize liver lysosomesin vitro. However, there is a contradictory finding by Robinson and Willcox (1969)who found that aspirin had no effect on liver lysosome stability in vitro. The fact that aspirin stabilizes retinal lysosomesagainst the labilizing effect of retinol in vitro suggeststhat if the retinal degeneration is mediated by the mechanism suggestedin the introduction, long-term administration of aspirin to dystrophic animals may retard the progress of the retinal degeneration in vivo. However, this
RELEASE
OF
LYSOSOMAL
ENZYMES
FROM
RAT
RETINA
71
may prove difficult to demonstrate in practice. In vitro results indicate that the desired stabilizing effect of aspirin only occurs over a comparatively narrow concentration range and to maintain the correct concentration of aspirin in the rat retina in vivo may well prove difficult judging from the known pharmacokinetic properties of aspirin (Rowland and Riegelman, 1968). Another consideration is that the labile visual pigment-retinol-lysosomal activation mechanism may just be one of a number of mechanisms involved in producing retinal degeneration in the dystrophic rats. In a study of retinal DNA levels in light deprived Campbell (albino dystrophic) rats we ha,ve demonstrated that deprivation of light until 8 weeks only retards, but does not abolish, the progress of the retinal degeneration (Dewar and Reading, 1974a). Ahhough the presence of pigment in the Hunter strain appears to slow down retinal degeneration (Yates et al., 1974) depriving these rats of light does not produce ati great an improvement as that seen in light-deprived Campbells (Dewar and Reading. 1974a). This suggests that a light-independent mechanism may be operative in addition to the light-dependent mechanism involving the labile visual pigment. Farber and Lolley (1973) have shown that in the dystrophic mouse (C3H strain), in addition to an opsin deficiency, there was a deficiency of a cyclic nucleotide phosphodiesterase. This occurs before onset of photo-receptor cell degeneration. We have preliminary evidence that, this is also true of the dystrophic rat (Dewar, Barron and Richmond, unpublished results). Such a deficiency would have a serious effect on the differentiation of photoreceptor cells by producing an abnormally high cyclic-AMP level in the retina (Lolley. Schmidt and Farber, 1974). Cyclic-AMP has been shown to have a labilizing effect on liver lysosomes(Imre? 1972) and if this is true of the retina one might expect the increased cyclic-AMP levels to aggravate any lysosome-labilizing effect of retinol. However, although aspirin merits further investigation as a possible agent for combating retinal degeneration, its usefulnessmay be limited by its pharmacokinetic properties and by the existence of other factors which cause degeneration
REFERENCES Bourne, M. C., Campbell, D. A. and Tansley, K. (1938). Hereditary degeneration of the rat retina. Brit. J. Ophthulnwl. 22, 613. Burden, E. M., Yates, C. M., Reading, H. W., Bitensky, L. and Chayen, J. (1971). Investigation into the structural integrity of lysosomes in the normal and dystrophic rat retina. Erp. E?ye Res. 12, 159. Chayen, J., Bitensky, L. and Ubhi, 0. S. (1972). The experimental modification of lysosomal dysfunction by anti-inflammatory drugs acting in vitro. Be&r. Path. Bd. 147,6. Dewar, A. J. and Reading, H. W. (1974a). The role of retinol in, and the action of anti-inflammatory drugs on, hereditary retinal degeneration. In Impaimnent of Cellular Functions during Age&g In Vivo and In Vitro. (Ed. Cristofolo,V. J.). PlenumPress,NewYork. Dewar, A. J. and Reading, H. W. (197413). Stabilization and labilization of lysosomes in rat retina. Biochemical Society Transactions 2, 645. Dowling, J. E. and Sidman, R. L. (1962). Inherited retinal dystrophy in the rat. J. CeZZ Biol. 14, 73. Farber, D. B. and Lolley, R. N. (1973). Proteins in the degenerative retina of C3H mice: deficiency of a cyclic-nucleotide phosphodiesterase and opsin. J. Neurochem. 21, 817. Fell, H. B., Dingle, J. T. and Webb, M. (1962). Studies on the mode of action of excess of vitamin A. 4: the specificity of the effect on embryonic chick limb cartilage in culture and on isolated rat liver lysosomes. Biochem. J. 83, 63.
72
A.
J.
DEWAR,
C:. BARRON
ASD
H.
W.
READISG
Imre, S. (1972). 3’5’-CAMP and lysosomal membrane labilization. h’xperientin, 28, 513. Lolley, R. N., Schmidt, 8. Y. and Farber, D. B. (1974). Alterations in cyclic AMP associated with photoreceptor cell degeneration in the C3H mouse. J. Seurochem. 22, 701. Mangla, J. C., Kim, Y. M. and Rubulis. A. A. (1974). Sdenyl cyclase stimulation by aspirin ir: rat gastric mucosa. Nature (London) 250, 61. Powell, E. D. U. and Field, R. A. (1964). Diabetic retinopathy and rheumatoid arthritis. Lmncet, ii, 17. Reading, H. W. (1966). Retinal and retinol metabolism in hereditary degeneration of the retina. Biochem. J. 100, 34P. Reading, H. W. (1970). Biochemistry of retinal dystrophy. J. IUecl. Genetics 7, 277. Robins, E., Fisher, K. and Lowe, I. P. (1961). Quantitative histochemical studies of the morphogenesis of the cerebellum. J. Neurochem.. 8, 96. Robinson, D. and Willcox, P. (1969). Interaction of salicylates with rat liver lysosomes. Biochem. J. 115,54P. Rowland, $1. and Riegelman, S. (1968). Pharmacokinetics of acetylsalicylic acid and salicylic acid after intravenous administration in man. J. Pharm. Xci. 57, 117. Schmidt, S. Y. and Lolley, R. N. (1973). Cyclic nucleotide phosphodiesterase: an early defect in inherited retinal degeneration of C3H mice. J. Cell Biol. 57, 117. Tanaka, K. and Lizuka, Y. (1968). Suppression of enzyme release from isolated rat liver lysosomes by non-steroidal anti-inflammatory drugs. Biochem. Pharmawl. 17, 2023. Yates, C. M., Dewar, A. J., Wilson, H., Winterburn, A. K. and Reading, H. W. (1974). Histological and biochemical studies on the retina of a new strain of dystrophic rat. Exp,. Eye Res. 18, 119. Vento, R. and Cacioppo, F. (1973). The effect of retinol on t’he lysosomal enzymes of bovine retinae and pigment epithelium. Exp. Eye Res. l&43.
The Effect of Retinol and Acetylsalicylic Acid on the Release of Lysosomal Enzymes from Rat Retina in vitro A. J; DEWAR, GILLIAN
BARRON AND H. W. READING
MRC Brain Metabolism Unit, Department of Pharmacology, University of Edinburgll, 1 GeorgeSquaw, Edinburgh, EH8 9JZ, U.K. (Received18 September1974, London) Retinol caused an increased release of fi-glucuronidase, ,&galartosidase and hexosaminidase from rat retinal lysosomes. Maximal labilization was produced by 1.5 rg/mg wet weight of retinal tissue. Acetylsalicylic acid in concentrations of 0*25-0.50 mM produced a significant stabilizing effect on rat retinal lysosomes both in the absence and presence of 1.5 pg/mg of retinol. It is suggested that acetylsalicylic acid and other anti-inflammatory drugs may merit further investigation as possible agents for combating retinal degeneration.
1. Introduction Degeneration of the retina in the strain of albino dystrophic rats known asCampbells (Bourne, Campbell and Tansley, 1938), is associatedwith an increase in the level of “free” lytic enzymeswhich appear to originate from lysosomesin the adjacent pigment epithelium (Burden, Yates, Reading, Bitensky and Chayen, 1971). It has been suggested (Reading, 1970) that the degeneration of the visual cells is produced by breakdown of lysosomal membranesin pigment epithelium and retina. This breakdown is thought to be due to an abnormal build-up of vitamin A (retinol) in the pigment epithelium which arisesfrom the action of light on an unusually labile type of visual pigment (Reading, 1966). It has been demonstrated that depriving Campbell rats of light, significantly retards the progress of the retinal degeneration (Dowling and Sidman, 1962; Dewar and Reading, 1974a). The presence of pigment in the eye in rats of a pigmented dystrophic strain known as Hunters also retards the progress of the degeneration (Yates, Dewar, Wilson, Winterburn and Reading, 1974). It has been demonstrated that retinol labilizes lysosomesin a variety of tissues (Fell, Dingle and Webb, 1962) and recently Vento and Cacioppo (1973) described the increasedreleaseof lysosomalenzyme produced by retinol in bovine retina and pigment epithehum in vitro. There is evidence that anti-inflammatory agents have a stabilizing effect on lysosomesand can retard the labilizing effects of histamine in inflammation (Chayen, Bitensky and Ubhi, 1972). In addition, a clinical study by Powell and Field (1964) revealed that a group of diabetic patients suffering from rheumatoid arthritis and requiring continuing large dosesof salicylates had a reduced incidence of retinopathy compared with the general diabetic population. This paper presents the results of a study of the effects of retinol and acetylsalicylic acid on rat retinal lysosomes. Part of this work has been presented as a preliminary communication (Dewar and Reading, 1974b). 2. Materials and Methods The e$ect of retinol on retinal
lysosomal stability
Sighted albino Wistar or Piebald Virol Glaxo (PVG) rats of either sex and of an age range I-3 months were used throughout. from both strains of rat.
Similar 63
results were obtained
with the retinas
6-l
A. J. DEIVAII,
c:. AAI’,KOS ASI) Il. IT. REAlJIx’C;
The rats were killed by tlecapitation. Retinas were removed aud homogenixetl iI1 12 url ice cold 0.25 &I-sucrose/g wet weight retina tissue for (1.5 min at ~OOO rev/nliu. The prc~cipitates were discarded and the superuatant centrifuged for two further .‘)-nlitl period% at IICUOg. The supernntant was t,heu split into 0.5 ml irliquots (eac:h containing the equivaletit of 46 nig of original tissue) arid these were added to tubes couta,inillg 293 ml (I.d5 M-sucrose plus kither O.l(y” V/V Triton Y-lCJ0 or O-2.5 pg/mg origiual tissue retinal. The retinol was dissolved in the minimutu volume of ethauol. This volume of ethanol was al+o added to the tubes not containing retinol. In most experiments all tubes were incubwtetl at 37°C for 45 min under a continuous flow of nitrogen but in some, the incubation time was varied from O--l80 min. After incubntion, each tube was cooled t.o (1°C ant1 t,he contents then centrifuged at 15 000 g for 20 min to pellet the intact lysosomes. To nsse”& the damage to the incubated lysoaomes the activities of four lysoeomnl enzymes: P-glucuronidase, acid phosphntase, ,&galactosidase and hexosaminidase mere measured. All activities were expressed as a percentage of the total releasable activity, i.e. as the percentage of the activity in the supernutaut derived from the tubes treated with 0.1 % Triton S-100.
/3-G’luczcro~l.idnse. /SGlucuronidase activity was measured by incubating 1 ml of each lysosomal-free supernatant with 0.2 ml 0.1 ;\I-phenolphthalein-mono-p-glucuronate (Sigma) in 1.6 ml 0.1 M-sodium acetate buffer, pH 4.5 for 19 hr at 37°C. Prior experiments had confirmed that the reaction proceeded in a linear manner over this period. At the end of the incubation protein was precipitated by the addition of 1 ml Sgb trichloracetic acid. Two ml 0.4 M-glycine buffer (pH 10.45) were added to 2 ml of the protein-free supernatant. After 30 min the extinction at 540 nm was determined. This method was derived from that, of Vento sud Cacioppo (1973). Acid phospl~atase. Acid phosphatase was supernatnnt to 1 ml citrate buffer, pH 5.5 which had been preincubated for 10 min at and 45 min incubation at 37°C by addition measured spectrophotometrically at 400 nm.
assayed by adding l-ml aliquots of diluted containing 0.1 M-paranitrophenyl phosphate 37°C. The reaction was stopped after 15, 30 of 1 ml 0.5 RI-NaOH. The p-nitrophenol was
/SGulactosidase. p-Galactosidase was measured by adding 0.5 ml of supernatant to 1 ml of 0.1 &I-glycine-HCl buffer pH3 containing 1 x 10~~ af-4-methylun~belliferylgalactosicle (MUG) plus 0.5 ml distilled water which had been preincubated at 37°C for 10 min. After incubation at 37°C for 60 min, 2 ml 0.1 H-Na,CO, was added and the fluorescence read at an excitation wavelength of 365 nm and an emission wavelength of 450 nm on a PerkinElmer spectrophotofluorimeter. This method is an adaptation of that of Yates (1973, Personal Communication) for measuring /%galactosidase in rat brain. This, in turn, was a modification of the method of Robins, Fischer and Lowe (1961). Hexosaminidase. Hexosaminidase was measured by adding 0.2 ml of the supernatant to 0.8 ml of 2.5 mM-4-methylumbe~iferyl-2-acetamido-2-dioxy-~-D-glucopyranoside in a buffer composed of 50 parts 0.2 M-Na,HPO, and 50 parts 0.1 M-citric acid. After 30 min incubation at 37°C the reaction was stopped by the addition of 3 ml 0.1 M-Na,CO,. The fluorescence was read at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. The effect of acetylsalicylic
acid on retinal lysosome
stability
The stability of lysosomes from normal retinas was examined in the presence of O-10 mMacetylsalicylic acid and in the presence of O-1 mM-aCetyhdiCyh acid plus 1.5 pg/mg original tissue retinol. The methods of preparation, incubation and enzyme assay were identical to those described above.
RELEASE
OF
LYSOSOMAL
ENZYMES
FROM
RAT
RETINA
65
The effect of retinol and acetylsalicylic acid on the activity of released lysosomal enzymes Retinal lysosomes were prepared and incubated for 45 min at 3’7% with 0.1% v/v Triton X-100 to release the lysosomal enzymes. After centrifuging the cooled incubation mixture at 15 000 g for 20 min, the supernatant was removed and split into three portions. One portion was used to test the effect of a range of acetylsahcylic acid concentrations on the activities of fi-glucuronidase, /Lgalactosidase, hexosaminidase and acid phosphatase, a second was used to test the effect of a range of retinol concentrations, and the third portion was used to determine the effect of a combination of acetylsalicylic acid and retinol.
3. Results The effect of retimol on lysosomal stability Retinol significantly increased the release of /3-glucuronidase, hexosaminidase and p-galactosidase from retinal lysosomes (Fig. 1). An increased release of /Sglucuronidase was observed with all concentrations of retinol tested but in the case of pgalactosidase and hexosaminidase a significantly increased release was observed only with retinol concentrations of 1.0 pg/mg and above. In all three case maximal release was obtained with 1.5 pg/mg retinol in the incubation medium. 8
“0 8
0
05
IO Concentration
I.5 of retinal
2.0 pg/mg
2.5
wet weight
FIG. 1. The effect. of retinol on the release of enzymes from retinal lysosomes during a 45-min incubation at 37”~. Points represent mean + S.D. of the number of determinations shown in brackets. The levels of significance (t test) of the increased release produced by 15 pg/mg were: fl-glucuronidase t = 7.66. P ( O@Ol; hexosaminidase t = 4.35, P < 0.01: j3-galactosidase t = 6.42, P < 0.001. The increase in acid phosphatase release was not significant. ,%Glucuronidase, 0 - - - 0 ; acid phosphatase, x -x : p-galactosidase, 0 -0 ; hexosaminidase, l __ 0.
The retinol failed to have a significant effect on the releaseof acid phosphatasesince. even in the absence of retinal, the bulk of the lysosomal acid phosphatase had entered the supernatant fraction during the course of the 45-min incubation. It was possible that the increased lysosomal enzyme activity in the supernatant fraction after retinol treatment was not a releasephenomenonbut an activation of the releasedenzymes. However, the results presented in Fig. 2 suggestthat this is unlikely, Retinol appeared to have very little effect on the activities of the releasedenzymes E
66
A.
.c >r .5 z D
J.
DEW’AR,
G. BARROS
AED
H.
\2’. READIN(:
90 eo-
8 :: 3
70-
i$
50-
.2 .g
40-
=i
30
GO-
I 0.5
0
I I.0
Concentration
2. The effect of retinol duplicates. Acid phosphatase, glucuronidase, l - - - 0. FIG.
I 15
of retinol
pg/mg
on the activity of released x x ; hexosaminidase,
TABLE
I 20 original
retinal
l
2.5
tissue
lysosomes. Points represent __ l ; /3-galactosidase, 0 --
mean of 0 : ,%
I
The effect of acetylsalicylic acid on /%glucuronidasereleasefrom retilzal lysosomesduring a 45-m& incubation at 37°C Concentration ASA (mM) 0 (Control) 0.05 0.10 0.25 0.50 1.00 2.50 5.00 7.50 10.00
*P
< 0.01.
of
o!oRelease in absence of retinol 49.5 54.1 65.0 17.1 21.5 34.9 24.1 71.3 79.6 123.0
t P < 0.025. Values represent
* + i * & + & (2) (2) k
22 (14) 2.6 (3) 1.4 (3) 5.4 (5)* 11.5 (6)t 13.9 (3) 6.1 (3):
4.9 (3)
%Release with 1.5 pg/mg retinal in incubation medium 112.1 104.2 119.3 54.3 26.6 88.5 72.6 70.0 70.0 121.6
$ P < 0.05. § P < 0~0001. mean * S.U. n shown in brackets.
* * * * += & (2) (2) (2) f
94 33.2 14.7 9.2 15.9 8+3
(9) (3) (4) (4)s (6)s (4)**
1.08 (4) ** P < 0.005.
and certainly not enough to explain the results shown in Fig. 1. However, the fact that the release of P-glucuronidase produced by 1.5 pg/mg retinol slightly exceeds the theoretical 100% (Fig. 1) doessuggestthat retinol may slightly activate the enzyme as well as promoting its release.This is not borne out by the data in Fig. 2 but the possibility that the presence of Triton X-100 may abolish any activating effect of retinol cannot be excluded. The effect of acetylsalicylic acid on lysosomestability Acetylsalicylic acid at concentrations of 0.25-0.50 mM significantly reduced the releaseof all the lysosomal enzymes tested (Tables I and II). At concentrations less
86.3 87.3 82.6 47.9 61.0 93.5
0 0.05 0.10 0.25 0950 1.00
of retinol
f 15.1 (11) * 10.3 (4) * 27.7 (5) * 14.0 (ll)* * 16 (5) + 15.5 (4)
In absence
u/ORelease
Cm@
c011c. ASA
of acid
The e#ect of ncetylsalicylic
II
96.2 77.5 63.5 43.1 45.7 53.0
Values
-J= (2) (2) + & +
16.1
(5)
retinol
* P < 0.001. represent
6.6 (5)t 4.9 (5)$ 21.1 (3)
+ I.5 pg/mg
phosphatase
mean
& & & * + -+ 10.8 5.7 7.1 8.9 13.8 7.5
(8) (3) (4) (?I)* (S)$ (3)
or retinol
t P < 0.005. k S.D. where
41.3 46.3 41.7 11.8 22.4 50.6
In absence
~/oRelease
$ P < 0.05. appropriate.
78.4 53.0 44.0 13.1 23.2 45.3
& * f f + &
(5) (3)$ (3)s (4)* (4)* (3)*
retinol
ti shown
in brackets.
41.6 34.3 30.3 0.37 9.2 15.1
In absence
retinal
i & f + & *
5.4 (4) 4.7 (3) 1.9 (3)$ 0.44 (3)* 0.94 (3)* 0.47 (3)*
of retinol
%Release
release from
5 P < 0.025.
5.6 14.1 15.5 6.4 2.2 1.8
+ 1.5 pg/mg
of 8.gatactosidasc
n&d on rtcid phosphatase /3-galaetosidase and hexosamirkdase a 45min. incubation at 37°C
TABLE
71.6 68.0 73.6 19.0 19.0 39.3
* & 3 & & &
+ 1.5 pg/mg
of hexosaminidase
5.3 1.4 4.4 12.7 2.5 2.6
(3) (3) (3)s (3)s (3)t (3)s
retinol
lysosomes during
0 (Control) 0.05 0.10 0.25 0.50 1.00 5.00 10.00
_-__
Concentration acetylsalicylic acid (mu)
of
100 92 98 92 96 95 94 92
In absence retinol
___-
of +l+
100 97 92 85
92
f&w retinol
Acid
ret&al
108 100 102 103 92
the mean
Values
+ 1.5 pgbg retinol
of duplicates.
100 104 109 109 109 114 104 104
Activity
of
95 100 109 100 -
In absence retinol
100 106 109 106 100 102 106 106
of cm&-ok
of
102 98 96 -
99
retinal
-: I.5 pg/mg
Hexosaminidase
expressedas percentage
+ 1.5 pglmg retinol
,%Galactosidase
enzymes.
In absence retinol
lysosomal
111
100 102 104 105 104 101 99 90
represent,
of
phoaphatase
of released
In absence retinol
acid on the activity
p-Glucuronidase
The e$ect of ucetylsalicylic
TABLE
RELEASE
OF
LYSOSOMAL
ENZYMES
FROM
RAT
RETINA
69
than 0.10 mM it had no effect except on hexosaminidase release. The release of /3glucuronidase was reduced by acetylsalicylic acid concentrations as high as 2.5 mM hut at higher concentrations it appeared to have a labilizing effect. Acetylsalicylic acid significantly stabilized retinal lysosomes against the labilizing action of 1.5 pg/mg retinol. In the absence of acetylsalicylic acid retinol produced an enzyme release ranging from 78 to ll2o/o but in the presence of the optimum concentration of acetylsalicylic acid this was reduced to between 13.1 and 43.1%. In the case of /3-glucuronidase release 0.25-7.50 mM had a stabilizing effect but the maximal effect was produced by 0.50 mM. At concentrations in excess of 7.5 mM there was no stabilizing effect. In the case of the other lysosomal enzymes, 0.25 mM-acetylsalicylic acid appeared to be the most effective stabilizing concentration. Acetylsalicylic acid had little effect on the activity of lysosomal enzymes after their release (Table III) thus indicating that its effect is to stabilize retinal lysosomes rather than inhibit the activity of the enzymes after release.
-_ _0 Preparation and centrifugation
30
45
120
60 Duration
of incubation
180
(mid
FIG. 3. The effect of incubation time on the effects of Triton X-100, retinol and acetylsalicylic acid on retinal lysosomal enzyme release. Points represent mean (f S.D. where applicable) of the number of determinations shown in brackets. (a) Hexosaminidase, Triton X-100, 0 l ; 1.5 pg/mg retinal, m---m; noadditions, A.... .A; 1.5 pg/mg retinol + 0.26 mu-acetylaalicylic acid, n---A ; 0.25 mM-acetylsalicylic acid, 0 -0. (b) p-Galactosidase. Triton X-100, 0 __ 0 ; 1.5 pg/mg retinal, m---w;noadditions,A.... . A; 0.25 m&x-acetylsalicylic acid. 0 __ 0 ; 0.25 mM-acetylsalicylic acid, A-/J.
io
A. J.
DEWAR,
Q.
BARROK
AND
H.
W.
READISC:
The e$ect of imxbation time on, the effect of Tritocn-X 100, retinol and acetylsalicylic acid lysosome stability This wasinvestigated usingP-galactosidaseand hexosaminidaseas enzyme markers. The results are shown in Fig. 3. Triton X-100 (0.1%) causedmaximum releaseof these enzymes within 45 min. During incubations exceeding 1 hr in duration, the Triton X-100 releasedactivity fell, presumably due to a direct inhibitory effect of Triton X-100 on the enzyme activity after release.A similar drop in activity was seenin the incubations with 1.5 pg/mg retinol. The lysosomal stabilizing effect of 0.25 mMacetylsalicylic (both in the presence and absenceof 1.5 pglmg retinol) apppeared to be maintained during a 3 hr incubation at 37°C. There are marked differences in the enzyme activity releasedin the presenceof Triton X-100, retinol and acetylsalicylic acid even in the absenceof an incubation at 37°C. This indicates that these agents affect lysosomestability during the centrifugation at 15 000 g. on retinal
4. Discussion We have demonstrated that retinol has a profound labilizing effect on rat retinal lysosomes.This is in agreement with the findings of Vento and Cacioppo (1973) who found that 1.5 pg/mg retinol produced the maximum labilizing effect on bovine retinal lysosomes.However, Vento and Cacioppo considered that retinol had a considerable activating effect on /3-glucuronidasesince the releasedactivity of this enzyme was as much as 28Oo/oof the activity releasedby sonication. The hypothesis outlined in the introduction suggeststhat the elevated retinol levels in the pigment epithelium of the dpstrophic rat results in an increased release of lysosomal enzymes. The present results demonstrate that retinol does labilize lysosomesbut doessoin concentrations considerably larger than the retinol levels reported by Reading (1966, 1970) who found that the tissue retinol level of the pigmented layers of 2-week-old dystrophic rats was ll-lSpg/g wet weight and in normal sighted rats approximately 7 lug/g wet weight. However, these absolute in vivo levels are almost certainly an underestimate. The pigment layers used in the assay consisted of pigment epithelium, choroid and sclera (i.e. the back of the eye minus the retina) and consequently the figures obtained must have been subject to oonsiderable dilution due to the presenceof excesstissue. In addition the pigment layers sample did not include the extralamellar material from the dystrophic rats. Thus at present it is impossible to say with certainty whether the retinol effect we observe unequivocally in vitro alsooccurs in vivo in the dystrophic retina. However, it is probable that the retina and the extralamellar material of the dystrophic rats contain far higher concentrations of retinol than the pigment epithelium and that local concentrations of retinol in the retina are sufficient to labilize lysosomes. Our results indicate that acetylsalicylic acid significantly stabilizes retinal lysosomes at certain concentrations but labilizes them at higher concentrations. The optimum stabilizing concentrations found in our experiments was the same as that reported by Tanaka and Lizuka (1968) to stabilize liver lysosomesin vitro. However, there is a contradictory finding by Robinson and Willcox (1969)who found that aspirin had no effect on liver lysosome stability in vitro. The fact that aspirin stabilizes retinal lysosomesagainst the labilizing effect of retinol in vitro suggeststhat if the retinal degeneration is mediated by the mechanism suggestedin the introduction, long-term administration of aspirin to dystrophic animals may retard the progress of the retinal degeneration in vivo. However, this
RELEASE
OF
LYSOSOMAL
ENZYMES
FROM
RAT
RETINA
71
may prove difficult to demonstrate in practice. In vitro results indicate that the desired stabilizing effect of aspirin only occurs over a comparatively narrow concentration range and to maintain the correct concentration of aspirin in the rat retina in vivo may well prove difficult judging from the known pharmacokinetic properties of aspirin (Rowland and Riegelman, 1968). Another consideration is that the labile visual pigment-retinol-lysosomal activation mechanism may just be one of a number of mechanisms involved in producing retinal degeneration in the dystrophic rats. In a study of retinal DNA levels in light deprived Campbell (albino dystrophic) rats we ha,ve demonstrated that deprivation of light until 8 weeks only retards, but does not abolish, the progress of the retinal degeneration (Dewar and Reading, 1974a). Ahhough the presence of pigment in the Hunter strain appears to slow down retinal degeneration (Yates et al., 1974) depriving these rats of light does not produce ati great an improvement as that seen in light-deprived Campbells (Dewar and Reading. 1974a). This suggests that a light-independent mechanism may be operative in addition to the light-dependent mechanism involving the labile visual pigment. Farber and Lolley (1973) have shown that in the dystrophic mouse (C3H strain), in addition to an opsin deficiency, there was a deficiency of a cyclic nucleotide phosphodiesterase. This occurs before onset of photo-receptor cell degeneration. We have preliminary evidence that, this is also true of the dystrophic rat (Dewar, Barron and Richmond, unpublished results). Such a deficiency would have a serious effect on the differentiation of photoreceptor cells by producing an abnormally high cyclic-AMP level in the retina (Lolley. Schmidt and Farber, 1974). Cyclic-AMP has been shown to have a labilizing effect on liver lysosomes(Imre? 1972) and if this is true of the retina one might expect the increased cyclic-AMP levels to aggravate any lysosome-labilizing effect of retinol. However, although aspirin merits further investigation as a possible agent for combating retinal degeneration, its usefulnessmay be limited by its pharmacokinetic properties and by the existence of other factors which cause degeneration
REFERENCES Bourne, M. C., Campbell, D. A. and Tansley, K. (1938). Hereditary degeneration of the rat retina. Brit. J. Ophthulnwl. 22, 613. Burden, E. M., Yates, C. M., Reading, H. W., Bitensky, L. and Chayen, J. (1971). Investigation into the structural integrity of lysosomes in the normal and dystrophic rat retina. Erp. E?ye Res. 12, 159. Chayen, J., Bitensky, L. and Ubhi, 0. S. (1972). The experimental modification of lysosomal dysfunction by anti-inflammatory drugs acting in vitro. Be&r. Path. Bd. 147,6. Dewar, A. J. and Reading, H. W. (1974a). The role of retinol in, and the action of anti-inflammatory drugs on, hereditary retinal degeneration. In Impaimnent of Cellular Functions during Age&g In Vivo and In Vitro. (Ed. Cristofolo,V. J.). PlenumPress,NewYork. Dewar, A. J. and Reading, H. W. (197413). Stabilization and labilization of lysosomes in rat retina. Biochemical Society Transactions 2, 645. Dowling, J. E. and Sidman, R. L. (1962). Inherited retinal dystrophy in the rat. J. CeZZ Biol. 14, 73. Farber, D. B. and Lolley, R. N. (1973). Proteins in the degenerative retina of C3H mice: deficiency of a cyclic-nucleotide phosphodiesterase and opsin. J. Neurochem. 21, 817. Fell, H. B., Dingle, J. T. and Webb, M. (1962). Studies on the mode of action of excess of vitamin A. 4: the specificity of the effect on embryonic chick limb cartilage in culture and on isolated rat liver lysosomes. Biochem. J. 83, 63.
72
A.
J.
DEWAR,
C:. BARRON
ASD
H.
W.
READISG
Imre, S. (1972). 3’5’-CAMP and lysosomal membrane labilization. h’xperientin, 28, 513. Lolley, R. N., Schmidt, 8. Y. and Farber, D. B. (1974). Alterations in cyclic AMP associated with photoreceptor cell degeneration in the C3H mouse. J. Seurochem. 22, 701. Mangla, J. C., Kim, Y. M. and Rubulis. A. A. (1974). Sdenyl cyclase stimulation by aspirin ir: rat gastric mucosa. Nature (London) 250, 61. Powell, E. D. U. and Field, R. A. (1964). Diabetic retinopathy and rheumatoid arthritis. Lmncet, ii, 17. Reading, H. W. (1966). Retinal and retinol metabolism in hereditary degeneration of the retina. Biochem. J. 100, 34P. Reading, H. W. (1970). Biochemistry of retinal dystrophy. J. IUecl. Genetics 7, 277. Robins, E., Fisher, K. and Lowe, I. P. (1961). Quantitative histochemical studies of the morphogenesis of the cerebellum. J. Neurochem.. 8, 96. Robinson, D. and Willcox, P. (1969). Interaction of salicylates with rat liver lysosomes. Biochem. J. 115,54P. Rowland, $1. and Riegelman, S. (1968). Pharmacokinetics of acetylsalicylic acid and salicylic acid after intravenous administration in man. J. Pharm. Xci. 57, 117. Schmidt, S. Y. and Lolley, R. N. (1973). Cyclic nucleotide phosphodiesterase: an early defect in inherited retinal degeneration of C3H mice. J. Cell Biol. 57, 117. Tanaka, K. and Lizuka, Y. (1968). Suppression of enzyme release from isolated rat liver lysosomes by non-steroidal anti-inflammatory drugs. Biochem. Pharmawl. 17, 2023. Yates, C. M., Dewar, A. J., Wilson, H., Winterburn, A. K. and Reading, H. W. (1974). Histological and biochemical studies on the retina of a new strain of dystrophic rat. Exp,. Eye Res. 18, 119. Vento, R. and Cacioppo, F. (1973). The effect of retinol on t’he lysosomal enzymes of bovine retinae and pigment epithelium. Exp. Eye Res. l&43.
