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The effect of retinol on the lysosomal enzymes of bovine retina and pigment epithelium
Ezp. Eye Res. (1973) 15, 4349
The Effect of Retinol on the Lysosomal Enzymes of Bovine Retina and Pigment Epithelium RENZA
VENTO
AND
FILIPPO
CACIOPPO
Istituto di Chimica Biologica dell’Univers&i (Received 16 March 1972
and
in
revised
form
di Palermo, Italy 31 August, 1972, Boston)
Retinal and pigment epithelium lysosomes, prepared from calf eyes, were obtained by fractional centrifugation. Free and total phosphatases, glucuronidases and proteases were studied by comparison with the corresponding activities released through the action of retinol. In vitro experiments indicate that incubation in the presence of retinol caused serious damage of the lysosomes from retina and pigment epithelium.
1. Introduction Studies carried out by Mellanby (1934, 1937, 1938, 1941) and Mellanby and Fell (1953) showed that retinol plays an important role in regulating the stability of cellular and subcellular membranes. Lucy and Dingle (1964) furthermore observed that small amounts of retinol will stabilize erythrocyte membranes, whereas large amounts alter them. On the other hand, when a rat-liver “mitochondrial” fraction is incubated with different amounts of retinol, the latter will induce the release of proportional amounts of lysosomal proteases (Dingle, 1961). The effect of retinol derivatives on the release of rat-liver lysosomal cathepsins and embryo chick limb cartilage was studied by Fell, Dingle and Webb (1962). They were able to show that only two of the substances examined were active : retinol and retinoic acid (the latter only when dissolved in ethanol). Administration of large amounts of retinol to rabbits (Thomas, McLuskey, Potter and Weissman, 1960) will produce alteration of the cartilage matrix, together with increased chondroitin sulfate contents of serum. Furthermore, when excess retinol is administered to adult rabbits (Weissman and Thomas, 1963) the result is an increased release of liver acid hydrolase. Dingle (1963) noticed that if chick embryo lysosomes are kept in contact with retinol, they release cathepsins. On the other hand, addition of retinol to a “mitochondrial” fraction results in increased release of lysosomal phosphatases into the incubation medium (De Duve, Wattiaux and Wibo, 1962) while, in vivo, hypervitaminosis A has similar effects on lysosomes (Dingle, Sharman and Moore, 1963). Weissman, Uhr and Thomas (1963), again reported an increased release of /3glucuronidase from the liver lysosomes of guinea pigs subjected to hypervitaminosis A, together with a very abundant quantity of /3-glucuronidase in the serum. We undertook the investigation of in vitro effects of retinol on cattle retinal and pigment epithelium lysosomes, in order to find out whether their reaction is similar to that already reported for lysosomes from other tissues. 2. Materials and Methods Reagents Tycosine, phenolphtalein mono$-glucuronic acid (sodium salt) and tram+retinol were supplied by Sigma Chemical Co. ; disodiumphenylphosphate by Riedel De Haenog Seelze, Hannover ; Folin-Ciocalteu reagent by Schmid et Co, Chroma Gesellschaft; bovine 43
44
R. VENT0
hemoglobine by Fluka (1 mg/O*l ml).
A. G., chemische
AND
F. CACIOPPO
fabrik.
Trans
retinol
was dissolved
in ethanol
Tissue preparatiovb Retinal and pigment epithelium lysosomes were prepared from calf eyes. All operations were carried out at 2-5°C. The retinae were gently rinsed with 0.25 M sucrose and homogenized in the same medium, in the ratio of 1 : 10 w/v, with a Potter Elvehjem homogenizer for 3 min at 1000 rev/min. The pigment epithelium was collected, after removing the retina, by carefully washing the inside at the eyeballs with small amounts of 0.25 M sucrose. The cell suspension was then spun for 10 min at 7000 g, the supernatant discarded and the resulting pellet weighed. The pigment epithelium pellet was then homogenized in a Potter-Elvehjem in a 0.25 M sucrose solution, in the ratio of 1 : 7 w/v for 3 min at 1000 rev/min. The retinal and pigment epithelium lysosomes were prepared by exactly the same procedure. The homogenate was spun for 5 min at 1100 g, the sediment discarded, and the resulting supernatant spun again at 1100 g for another 5 min ; this operation was repeated until no more pigment epithelium or cells were seen to separate (4 or 5 times were found to be sufficient). The supernatant was spun for 15 min at 10,000 g. After centrifugation, the supernatant was discarded, carefully avoiding to remove the flocculent layer which rested on top of the compact sediment. The latter was resuspended in 0.25 M sucrose and spun again for 5 min at 1100 g, so as to remove any pigment residue. The supernatant was finally spun for 10 min at 11,000 g and the resulting sediment, containing mitochondria and lysosomes (“lysosomal” fraction), was used for our research. Enzymic
assays
Enzyme assays included free enzymes, total enzymes and enzymes released through the action of retinol. Free enzymes (enzymes present in the “lysosomal” fraction due to damage of the lysosomes following preparation) were determined in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min at 15,000 g at O’C. Total enzyme activity was determined in the supernatant of the “lysosomal” fraction, after treatment for 5 min with an MSE Mullard ultrasonic disintegrator (20 kHz), followed by oentrifugation for 20 min at 15,000 g. For determination of the enzymes released through the action of retinol, the “lysosomal” fraction was incubated for 45 min at 37°C under nitrogen gas, and retinol was added in an amount of about 1.5 pg/mg original tissue. The preparation was then spun for 20 min at 15,900 g. The release of enzymes by the action of retinol was expressed as percentage of the total amount released after ultrasonic treatment. Free as well as total enzymic activities were determined after maintaining the respective “lysosomal” fractions under nitrogen gas for 45 min at 37”C, so the experimental conditions would be the same as for the retinol induced enzymic activities. Cathepsins These were determined by a modification of the method described by Anson (1938). The incubation mixture was made of 3 ml 0.1 M-CH,---COONa (adjusted to pH 4.5 with 0.1 M-CHaCOOH), also containing 2% hemoglobin, and 2 ml of the supernatant from the “lysosomal” fraction, after centrifugation for 20 min at 15,000 g at 0°C (approximately 650 mg of the original retinal tissue and 350 mg of the original pigment epithelium). The mixture was incubated for 1 hr at 37”C, 1 ml samples being removed every 10 min. At the end of the incubation period, protein separation was effected by adding to each sample 1 ml of 5% w/v trichloroacetic acid. Free tyrosine and tyrosine content of the peptides were determined, at the same time, by addition of 4 ml of 0.4 M NaOH and 1.2 ml Folin-Ciocalteu reagent (diluted 1 : 3 v/v with redistilled water). Adsorption at 540 nm was read after 20 min against standard concentrations of pure tyrosine, ranging from 0.02 to 0.2 micromoles in 0.06 M HCl.
RETINAL
AND
PIGMENT
EPITHELIUM
LYSOSOMES
45
Acid phosphatase The incubation mixture for the determination of acid phosphatases was made of 5 ml 0.2 M CHs-COONa (adjusted to pH 4.5 with O-2 M CHs-COOH), also containing 110 mg y0 of disodiumphenylphosphate, and 1 ml of the supernatant from the “lysosomal” fraction, after centrifugation at 15,000 g (approximately 300 mg of the original retinal tissue and 170 mg of the original pigment epithelium). The test tubes were maintained for 45 min at 37°C and 2 ml samples were removed every 15 min. At the end of the required incubation period, the enzymic reaction was stopped by adding to each test tube 4.5 ml Folin-Ciocalteu reagent (diluted 1 : 3 v/v with redistilled water), 8 ml redistilled water and 2.5 ml of 20% w/v Na,CO,. Absorption was read at 640 nm after 10 min against standard concentrations of phenol, ranging from 5 to 20 pg. Glucuronklase
The incubation mixture for the determination of glucuronidase activity consisted of 1.6 ml 0.1 M CH,-COONa (adjusted to pH 4.5 with 0.1 M CHs-COOH), O-2 ml of O-1 M phenolphtalein mono-/3-glucuronic acid and 1 ml of the supernatant from the “lysosomal” fraction, after centrifugation at 15,000 g (app roximately 500 mg of the original retinal tissue and 350 mg of the original pigment epithelium). Incubation was carried out at 37°C for periods of 1 to 24 hr. At the end of each period, separation of proteins was effected by adding to each test tube 1 ml of 5% w/v trichloroacetic acid. Displaced phenolphtalein was measured after mixing 2 ml of the supernatant with 2 ml 0.4 M glycine (adjusted to pH 10.45 with 0.4 M NaOH) and 0.5 ml of 0.5 JI NaOH. Readings were taken at 540 nm after 30 min.
05
23
I Retfnol
(pg/mg
of tissue)
Fm. 1. Effect of different concentrations of retinol on the release of enzymes from retinal and pigment epithelium “lysosomal” fraction. The “lysosomal” fraction of the retina and pigment epithelium were suspended in 0.26 rd-sucrose then incubated for 46 min at 37”C, under nitrogen, with different ooncentrations of retinol. At the end of the incubation all suspensions were spun for 20 min at 15,000 g at 0°C. Enzymic activities were determined in the supernatants and the amounts found were expressed as % of the total activity released by ultrasonic treatment. 0, proteolytic activities of pigment epithelium; a, acid phosphatase activities of pigment epithelium; 0, acid phosphatase activities of the retina; l , proteolytic aativities of the retina; A, ,$glucuronidase activities of the retina.
46
R. VENT0
AND
F. CACIOPPO
3. Results Effect of different concentrations of retinal Figure 1 shows the action of different concentrations of retinol on “lysosomal’ ’ suspensions of cattle retinal and pigment epithelium. Retinol exhibits similar effects on both types of “lysosomal” suspensions and its action is dependent on its concentration in all the types of enzymic lysosomal activity studied. Release of the proteases As shown in Table I, cathepsins are present in high amounts both in the retina and in the pigment epithelium preparations. As much as 1.5 pg of retinol/mg of the retinal tissue was sufficient to release this enzymic activity and their presence acts both on the retinal lysosomes and on those of the epithelium pigment releasing about 120% of the total activity (Fig. 2). Incubation for 40 min at 37°C was found to be the most favourable condition for testing protease activity. TABLE
I
Effect of retinol upon the release of enzymic activities from retinal and pigment epithelium “lysosomal” fraction Acid phosphatases
Free enzyme activity in retina Free enzyme activity in pigment Total enzyme activity in retina Total enzyme activity in pigment Retinol induced enzyme activity Retinol induced enzyme activity pigment epithelium
epithelium epithelium in retina in
Cathepsins
j3-glucuronidase
3.7 2.9 18.6 12.7 9.2
8.7 6.2 28.0 21.0 33.0
none none 1.4 none 4.8
6.2
28.0
none
Retinal and pigment epithelium “lysosomal” fraction were suspended in 0.25 ~-sucrose and incubated for 45 min at 37”C, under nitrogen gas, with or without retinol (15 pg/mg of tissue). At the end of the incubation period, the suspensions were spun for 20 min at 15,000 g at 0°C and acid phosphatases, cathepsins and /3-glucuronidase activities were measured in the supernatants while varying incubation periods. “Free enzyme activity” is the activity present in the “lysosomal” fraction due to damage of the lysosomes following preparation. “Total enzyme activity” is the activity obtained after ultrasonic treatment for 5 min of “lyso8omaI” fraction. “Retinol induced enzyme activity” is the activity obtained after incubation of “lysosomal” fraction with retinal. “Acid phosphatase” activities were expressed in terms of a unit arbitrarily defined as the activity, contained in 0.33 ml samples of supernatnant from the “lysosomal” fraction, which is able to set free 1 pg phenol from 1.66 ml of 5x 1OV M disodiumphenylphosphate, within 40 min, at a temperature of 37°C ,. “Protease” activities were expressed in terms of a unit arbitrarily defined as the enzymic activity, contained in 1 ml samples of supernatant from “lysosomal” fraction, which is able to set free, 10 pg of acid soluble tyrosine from 1.5 ml of 2% hemoglobin solution in 40 min at 37”C!. “8-glucuronidase” activity was expressed in terms of a unit arbitrarily defined as the enzymic activity, contained in 1 ml samples of aupernatant from the “lysosomal” fraction, which is able to set free 1 pg of phenolphtalein from 0.2 ml of 10-Z M phenolphtalein+-glucuronide in 3 hr at 37’C.
Acid phosphatase activity Also phosphatase activity is present in high levels both in retinal lysosomes and in those from pigment epithelium (Table I). The presence of retinol brings about the
RETINAL
AND
PIGMENT
EPITHELIUM
LYSOSOMES
47
release of approximately 60% of the activity found after ultrasonic treatment (Pig. 3). Incubation of the assay system for different periods of time shows these phosphatase activities to reach a peak when incubation at 37°C is protracted for 40 min.
20
40
60
Incubation
time
80
100
(min)
Fro. 2. Effect of retinol upon the release of cathepsins from retinal and pigment epithelium “lysosomal” fraction. Retinal and pigment epithelium “lysosomal” fractions were suspended in 0.25 M-sucrose and incubated for 45 min at 37”C, under nitrogen, with retinol (1.5 clg/mg tissue). At the end of the incubation period, the suspensions were spun for 20 min at 15,000 g at 0°C and proteolytic activities were measured in the supernatants while varying incubation periods. The amounts found were expressed as % of the total activity released by ultrasonic treatment. “Free cathepsins” were determined, after maintaining the “lysosomal” fractions under nitrogen for 45 min at 37”C, in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min at 15,000 g at 0°C. 0, free cathepsins; A, cathepsins released by retinol from pigment epithelium; 0, cathepsins released by retinol from retina.
G
20
40
60
Incubation
time
00
100
(min)
FIG 3. Release of acid phosphatases from retinal and pigment epithelium “lysosomal” fraction following treatment with retinol. Retinal and pigment epithelium “lysosomal” fraction were suspended in 0.25 M-sucrose and incubated with retinol (1.5 &mg tissue), under nitrogen, for 45 min at 37°C. When incubation was over, the suspensions were spun for 20 min at 15,000 g at 0°C and acid phosphetases were measured in the supernatante while varying incubation periods. The amounts found were expressed as oA of the total activity released by ultrasonic treatment. “Free phosphatases” were determined, after maintaining the “lysosomal” fraction under nitrogen for 45 min at 3’7”C, in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min released by retinol from pigment at 15,000g at 0°C. 0, free phosphatases; A, acid phosphatases epithelium; 0, acid phosphatases released by retinol from retina. D
48
R. VENT0
AND
F. CACIOPPO
Glucuronidase activity This activity is present in retinal “lysosomal” fraction. It was impossible to detect it in the same fraction obtained from pigment epithelium (Table I). However, the way retinol affects the release of this activity from retinal “lysosomal” fraction is quite surprising. After retinol is added to this fraction, the enzymic activity reaches as much as 350% of that obtained after ultrasonic treatment of this fraction. This is shown in Fig. 4. The investigation of this enzymic activity for different periods of incubation shows a gradual increase, until it reaches a maximum between the 17th and 24th hr.
bV
120 Incubation
180 time
240
300
(min)
FIG 4. Release of ,%glucuronidase from retinal “lysosomal” fraction. Suspensions of retinal “lysosomal” fraction in 0.25 M-sucrose containing retinol (15 pg/mg tissue) were incubated, under nitrogen, for 45 min at 37°C. The suspensions were spun for 20 min at 15,000 g at 0°C and ,%glucuronidase activity was measured in the supernatants while varying incubation periods. The amounts found were expressed as ‘A of the total activity released by ultrasonic treatment. “Free j3-glucuronidase” was determined, after maintaining the “lysosomal” fraction under nitrogen for 45 min at 37”C, in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min at 15,000 g at 0°C. 0, free /?-glucuronidase ; A, /J-glucuronidase released by retinol.
4. Discussion Our data shows that retinol is capable of penetrating the lysosomal membranes of bovine retina and pigment epithelium and by altering their stability causes release of lysosomal enzymes. The observation that the proteolitic and glucuronidase activities are released in the presence of retinol in percentage greater than that obtained after ultrasonic treatment, may be attributed to some enzyme activation process. Previous research has brought to light numerous conditions, both experimental and pathologic, in which an accumulation of retinol, or a series of conditions which might lead to it, takes place and is often accompanied by lysosomal damage. The amount of retinol measured in the pigment epithelium of young (24 weeks) rats affected by retinal dystrophy is at least double that at present in the pigment epithelium of normal rats (Reading, 1966). Amemiya (1967) has shown that, in animals in conditions of hypervitaminosis A, there are conspicuous changes in the outer segments and electron microscopic examination showed that the first changes take place in the pigment epithelium.
RETINAL
AND
PIGIbrIENT
EPITHELIUM
LYSOSOMES
49
In rats affected by retinal dystrophy, the rod outer segments are altered between the 12th and 14th days of age (Dowling, 1962) and the phagocytosis of the pigment epithelium of these animals are altered from the 15th day of age until it disappears (Herron, 1970). At the same age-period the lysosomes of the pigment epithelium of dystrophic rats show serious damage to their wall accompanied by the release of acid phosphatases, a damage which seems to precede retinal dystrophy (Yates, 1970). Our finding that retinol causes release and in some cases even activation of lysosomal enzymes in the retina and pigment epithelium in proportion to its concentration, has led us to assume that, in vivo, an accumulation of retinol might lead to similar result. If this would be the case in vivo, the inevitable consequence of the retinol effect on the release of lysosomal enzymes would be gradual retinal damage. Furthermore the normal lysosomal activity of the pigment epithelium, indispensable for the normal phagocytic activity of these cells, would also suffer. REFERENCES Amemiya, T. (1967). A& Sot. Ophtkal. Yap. 71, 2236. Anson, M. L. (1938). J. Gen. Physiol. 22, 79. De Duve, C., Wattiaux, R. and Wibo, M. (1962). Biochem. Pharmacol. 9, 97. Dingle, J. T. (1961). Biochem. J. 79, 509. Dingle, J. T. (1963). Ciba Foundation Symposium on Lysosomes, p. 384. Ed. by de Reuck, A. V. S. and Cameron, M. P. (Little, Brown and Co., Boston, Mass.). Dingle, J. T., Sharman, I. M. and Moore, T. (1963). Proc. Nut&. Sot. 22, X. Dowling, J. E. and Sidman, R. L. (1962). J. CeZ2 Biol. 14, 73. Fell, H. B. and Mellanby, E. (1953). J. Physiol. (London) 119,470. Fell, H. B., Dingle, J. T. and Webb, M. (1962). Biochem. J. 83,63. Herron, Warren, L., Byron W. Riegel, ORLOE., Myers and Melvin, L. Rubin. (1969). Invest. Opht?dnol. 8, 595. Lucy, J. A. and Dingle, J. T. (1964). Nature (London) 204, 156. Mellanby, E. (1934). J. Pa&Z. Bact. 38,391. Mellanby, E. (1937). Chem. Id (London) 56, 1054. Mellanby, E. (1938). J. Physiol. (London) 93,42. Mellanby, E. (1941). J. Physiol. (London) 99,467. Reading, H. W. (1966). B&hem. J. 100,34p. Thomas, L., McLuskey, R. T., Potter, J. L. and Weissman, G. (1960). J. Exp. Med. 111, 705. Weissman, G. and Thomas, L. (1963). J. Clin. Invest. 42, 661. Weissman, CT., Uhr, J. W. and Thomas, L. (1963). Proc. Sot. Exp. Biol. N. Y. 112, 284. Yates, M. C., Reading, H. W. Bitensky, L. and Chayen, J. (1970). Biochem. J. 119,57 p.
The Effect of Retinol on the Lysosomal Enzymes of Bovine Retina and Pigment Epithelium RENZA
VENTO
AND
FILIPPO
CACIOPPO
Istituto di Chimica Biologica dell’Univers&i (Received 16 March 1972
and
in
revised
form
di Palermo, Italy 31 August, 1972, Boston)
Retinal and pigment epithelium lysosomes, prepared from calf eyes, were obtained by fractional centrifugation. Free and total phosphatases, glucuronidases and proteases were studied by comparison with the corresponding activities released through the action of retinol. In vitro experiments indicate that incubation in the presence of retinol caused serious damage of the lysosomes from retina and pigment epithelium.
1. Introduction Studies carried out by Mellanby (1934, 1937, 1938, 1941) and Mellanby and Fell (1953) showed that retinol plays an important role in regulating the stability of cellular and subcellular membranes. Lucy and Dingle (1964) furthermore observed that small amounts of retinol will stabilize erythrocyte membranes, whereas large amounts alter them. On the other hand, when a rat-liver “mitochondrial” fraction is incubated with different amounts of retinol, the latter will induce the release of proportional amounts of lysosomal proteases (Dingle, 1961). The effect of retinol derivatives on the release of rat-liver lysosomal cathepsins and embryo chick limb cartilage was studied by Fell, Dingle and Webb (1962). They were able to show that only two of the substances examined were active : retinol and retinoic acid (the latter only when dissolved in ethanol). Administration of large amounts of retinol to rabbits (Thomas, McLuskey, Potter and Weissman, 1960) will produce alteration of the cartilage matrix, together with increased chondroitin sulfate contents of serum. Furthermore, when excess retinol is administered to adult rabbits (Weissman and Thomas, 1963) the result is an increased release of liver acid hydrolase. Dingle (1963) noticed that if chick embryo lysosomes are kept in contact with retinol, they release cathepsins. On the other hand, addition of retinol to a “mitochondrial” fraction results in increased release of lysosomal phosphatases into the incubation medium (De Duve, Wattiaux and Wibo, 1962) while, in vivo, hypervitaminosis A has similar effects on lysosomes (Dingle, Sharman and Moore, 1963). Weissman, Uhr and Thomas (1963), again reported an increased release of /3glucuronidase from the liver lysosomes of guinea pigs subjected to hypervitaminosis A, together with a very abundant quantity of /3-glucuronidase in the serum. We undertook the investigation of in vitro effects of retinol on cattle retinal and pigment epithelium lysosomes, in order to find out whether their reaction is similar to that already reported for lysosomes from other tissues. 2. Materials and Methods Reagents Tycosine, phenolphtalein mono$-glucuronic acid (sodium salt) and tram+retinol were supplied by Sigma Chemical Co. ; disodiumphenylphosphate by Riedel De Haenog Seelze, Hannover ; Folin-Ciocalteu reagent by Schmid et Co, Chroma Gesellschaft; bovine 43
44
R. VENT0
hemoglobine by Fluka (1 mg/O*l ml).
A. G., chemische
AND
F. CACIOPPO
fabrik.
Trans
retinol
was dissolved
in ethanol
Tissue preparatiovb Retinal and pigment epithelium lysosomes were prepared from calf eyes. All operations were carried out at 2-5°C. The retinae were gently rinsed with 0.25 M sucrose and homogenized in the same medium, in the ratio of 1 : 10 w/v, with a Potter Elvehjem homogenizer for 3 min at 1000 rev/min. The pigment epithelium was collected, after removing the retina, by carefully washing the inside at the eyeballs with small amounts of 0.25 M sucrose. The cell suspension was then spun for 10 min at 7000 g, the supernatant discarded and the resulting pellet weighed. The pigment epithelium pellet was then homogenized in a Potter-Elvehjem in a 0.25 M sucrose solution, in the ratio of 1 : 7 w/v for 3 min at 1000 rev/min. The retinal and pigment epithelium lysosomes were prepared by exactly the same procedure. The homogenate was spun for 5 min at 1100 g, the sediment discarded, and the resulting supernatant spun again at 1100 g for another 5 min ; this operation was repeated until no more pigment epithelium or cells were seen to separate (4 or 5 times were found to be sufficient). The supernatant was spun for 15 min at 10,000 g. After centrifugation, the supernatant was discarded, carefully avoiding to remove the flocculent layer which rested on top of the compact sediment. The latter was resuspended in 0.25 M sucrose and spun again for 5 min at 1100 g, so as to remove any pigment residue. The supernatant was finally spun for 10 min at 11,000 g and the resulting sediment, containing mitochondria and lysosomes (“lysosomal” fraction), was used for our research. Enzymic
assays
Enzyme assays included free enzymes, total enzymes and enzymes released through the action of retinol. Free enzymes (enzymes present in the “lysosomal” fraction due to damage of the lysosomes following preparation) were determined in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min at 15,000 g at O’C. Total enzyme activity was determined in the supernatant of the “lysosomal” fraction, after treatment for 5 min with an MSE Mullard ultrasonic disintegrator (20 kHz), followed by oentrifugation for 20 min at 15,000 g. For determination of the enzymes released through the action of retinol, the “lysosomal” fraction was incubated for 45 min at 37°C under nitrogen gas, and retinol was added in an amount of about 1.5 pg/mg original tissue. The preparation was then spun for 20 min at 15,900 g. The release of enzymes by the action of retinol was expressed as percentage of the total amount released after ultrasonic treatment. Free as well as total enzymic activities were determined after maintaining the respective “lysosomal” fractions under nitrogen gas for 45 min at 37”C, so the experimental conditions would be the same as for the retinol induced enzymic activities. Cathepsins These were determined by a modification of the method described by Anson (1938). The incubation mixture was made of 3 ml 0.1 M-CH,---COONa (adjusted to pH 4.5 with 0.1 M-CHaCOOH), also containing 2% hemoglobin, and 2 ml of the supernatant from the “lysosomal” fraction, after centrifugation for 20 min at 15,000 g at 0°C (approximately 650 mg of the original retinal tissue and 350 mg of the original pigment epithelium). The mixture was incubated for 1 hr at 37”C, 1 ml samples being removed every 10 min. At the end of the incubation period, protein separation was effected by adding to each sample 1 ml of 5% w/v trichloroacetic acid. Free tyrosine and tyrosine content of the peptides were determined, at the same time, by addition of 4 ml of 0.4 M NaOH and 1.2 ml Folin-Ciocalteu reagent (diluted 1 : 3 v/v with redistilled water). Adsorption at 540 nm was read after 20 min against standard concentrations of pure tyrosine, ranging from 0.02 to 0.2 micromoles in 0.06 M HCl.
RETINAL
AND
PIGMENT
EPITHELIUM
LYSOSOMES
45
Acid phosphatase The incubation mixture for the determination of acid phosphatases was made of 5 ml 0.2 M CHs-COONa (adjusted to pH 4.5 with O-2 M CHs-COOH), also containing 110 mg y0 of disodiumphenylphosphate, and 1 ml of the supernatant from the “lysosomal” fraction, after centrifugation at 15,000 g (approximately 300 mg of the original retinal tissue and 170 mg of the original pigment epithelium). The test tubes were maintained for 45 min at 37°C and 2 ml samples were removed every 15 min. At the end of the required incubation period, the enzymic reaction was stopped by adding to each test tube 4.5 ml Folin-Ciocalteu reagent (diluted 1 : 3 v/v with redistilled water), 8 ml redistilled water and 2.5 ml of 20% w/v Na,CO,. Absorption was read at 640 nm after 10 min against standard concentrations of phenol, ranging from 5 to 20 pg. Glucuronklase
The incubation mixture for the determination of glucuronidase activity consisted of 1.6 ml 0.1 M CH,-COONa (adjusted to pH 4.5 with 0.1 M CHs-COOH), O-2 ml of O-1 M phenolphtalein mono-/3-glucuronic acid and 1 ml of the supernatant from the “lysosomal” fraction, after centrifugation at 15,000 g (app roximately 500 mg of the original retinal tissue and 350 mg of the original pigment epithelium). Incubation was carried out at 37°C for periods of 1 to 24 hr. At the end of each period, separation of proteins was effected by adding to each test tube 1 ml of 5% w/v trichloroacetic acid. Displaced phenolphtalein was measured after mixing 2 ml of the supernatant with 2 ml 0.4 M glycine (adjusted to pH 10.45 with 0.4 M NaOH) and 0.5 ml of 0.5 JI NaOH. Readings were taken at 540 nm after 30 min.
05
23
I Retfnol
(pg/mg
of tissue)
Fm. 1. Effect of different concentrations of retinol on the release of enzymes from retinal and pigment epithelium “lysosomal” fraction. The “lysosomal” fraction of the retina and pigment epithelium were suspended in 0.26 rd-sucrose then incubated for 46 min at 37”C, under nitrogen, with different ooncentrations of retinol. At the end of the incubation all suspensions were spun for 20 min at 15,000 g at 0°C. Enzymic activities were determined in the supernatants and the amounts found were expressed as % of the total activity released by ultrasonic treatment. 0, proteolytic activities of pigment epithelium; a, acid phosphatase activities of pigment epithelium; 0, acid phosphatase activities of the retina; l , proteolytic aativities of the retina; A, ,$glucuronidase activities of the retina.
46
R. VENT0
AND
F. CACIOPPO
3. Results Effect of different concentrations of retinal Figure 1 shows the action of different concentrations of retinol on “lysosomal’ ’ suspensions of cattle retinal and pigment epithelium. Retinol exhibits similar effects on both types of “lysosomal” suspensions and its action is dependent on its concentration in all the types of enzymic lysosomal activity studied. Release of the proteases As shown in Table I, cathepsins are present in high amounts both in the retina and in the pigment epithelium preparations. As much as 1.5 pg of retinol/mg of the retinal tissue was sufficient to release this enzymic activity and their presence acts both on the retinal lysosomes and on those of the epithelium pigment releasing about 120% of the total activity (Fig. 2). Incubation for 40 min at 37°C was found to be the most favourable condition for testing protease activity. TABLE
I
Effect of retinol upon the release of enzymic activities from retinal and pigment epithelium “lysosomal” fraction Acid phosphatases
Free enzyme activity in retina Free enzyme activity in pigment Total enzyme activity in retina Total enzyme activity in pigment Retinol induced enzyme activity Retinol induced enzyme activity pigment epithelium
epithelium epithelium in retina in
Cathepsins
j3-glucuronidase
3.7 2.9 18.6 12.7 9.2
8.7 6.2 28.0 21.0 33.0
none none 1.4 none 4.8
6.2
28.0
none
Retinal and pigment epithelium “lysosomal” fraction were suspended in 0.25 ~-sucrose and incubated for 45 min at 37”C, under nitrogen gas, with or without retinol (15 pg/mg of tissue). At the end of the incubation period, the suspensions were spun for 20 min at 15,000 g at 0°C and acid phosphatases, cathepsins and /3-glucuronidase activities were measured in the supernatants while varying incubation periods. “Free enzyme activity” is the activity present in the “lysosomal” fraction due to damage of the lysosomes following preparation. “Total enzyme activity” is the activity obtained after ultrasonic treatment for 5 min of “lyso8omaI” fraction. “Retinol induced enzyme activity” is the activity obtained after incubation of “lysosomal” fraction with retinal. “Acid phosphatase” activities were expressed in terms of a unit arbitrarily defined as the activity, contained in 0.33 ml samples of supernatnant from the “lysosomal” fraction, which is able to set free 1 pg phenol from 1.66 ml of 5x 1OV M disodiumphenylphosphate, within 40 min, at a temperature of 37°C ,. “Protease” activities were expressed in terms of a unit arbitrarily defined as the enzymic activity, contained in 1 ml samples of supernatant from “lysosomal” fraction, which is able to set free, 10 pg of acid soluble tyrosine from 1.5 ml of 2% hemoglobin solution in 40 min at 37”C!. “8-glucuronidase” activity was expressed in terms of a unit arbitrarily defined as the enzymic activity, contained in 1 ml samples of aupernatant from the “lysosomal” fraction, which is able to set free 1 pg of phenolphtalein from 0.2 ml of 10-Z M phenolphtalein+-glucuronide in 3 hr at 37’C.
Acid phosphatase activity Also phosphatase activity is present in high levels both in retinal lysosomes and in those from pigment epithelium (Table I). The presence of retinol brings about the
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release of approximately 60% of the activity found after ultrasonic treatment (Pig. 3). Incubation of the assay system for different periods of time shows these phosphatase activities to reach a peak when incubation at 37°C is protracted for 40 min.
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Fro. 2. Effect of retinol upon the release of cathepsins from retinal and pigment epithelium “lysosomal” fraction. Retinal and pigment epithelium “lysosomal” fractions were suspended in 0.25 M-sucrose and incubated for 45 min at 37”C, under nitrogen, with retinol (1.5 clg/mg tissue). At the end of the incubation period, the suspensions were spun for 20 min at 15,000 g at 0°C and proteolytic activities were measured in the supernatants while varying incubation periods. The amounts found were expressed as % of the total activity released by ultrasonic treatment. “Free cathepsins” were determined, after maintaining the “lysosomal” fractions under nitrogen for 45 min at 37”C, in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min at 15,000 g at 0°C. 0, free cathepsins; A, cathepsins released by retinol from pigment epithelium; 0, cathepsins released by retinol from retina.
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FIG 3. Release of acid phosphatases from retinal and pigment epithelium “lysosomal” fraction following treatment with retinol. Retinal and pigment epithelium “lysosomal” fraction were suspended in 0.25 M-sucrose and incubated with retinol (1.5 &mg tissue), under nitrogen, for 45 min at 37°C. When incubation was over, the suspensions were spun for 20 min at 15,000 g at 0°C and acid phosphetases were measured in the supernatante while varying incubation periods. The amounts found were expressed as oA of the total activity released by ultrasonic treatment. “Free phosphatases” were determined, after maintaining the “lysosomal” fraction under nitrogen for 45 min at 3’7”C, in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min released by retinol from pigment at 15,000g at 0°C. 0, free phosphatases; A, acid phosphatases epithelium; 0, acid phosphatases released by retinol from retina. D
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Glucuronidase activity This activity is present in retinal “lysosomal” fraction. It was impossible to detect it in the same fraction obtained from pigment epithelium (Table I). However, the way retinol affects the release of this activity from retinal “lysosomal” fraction is quite surprising. After retinol is added to this fraction, the enzymic activity reaches as much as 350% of that obtained after ultrasonic treatment of this fraction. This is shown in Fig. 4. The investigation of this enzymic activity for different periods of incubation shows a gradual increase, until it reaches a maximum between the 17th and 24th hr.
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FIG 4. Release of ,%glucuronidase from retinal “lysosomal” fraction. Suspensions of retinal “lysosomal” fraction in 0.25 M-sucrose containing retinol (15 pg/mg tissue) were incubated, under nitrogen, for 45 min at 37°C. The suspensions were spun for 20 min at 15,000 g at 0°C and ,%glucuronidase activity was measured in the supernatants while varying incubation periods. The amounts found were expressed as ‘A of the total activity released by ultrasonic treatment. “Free j3-glucuronidase” was determined, after maintaining the “lysosomal” fraction under nitrogen for 45 min at 37”C, in the supernatant obtained from centrifugation of the “lysosomal” fraction for 20 min at 15,000 g at 0°C. 0, free /?-glucuronidase ; A, /J-glucuronidase released by retinol.
4. Discussion Our data shows that retinol is capable of penetrating the lysosomal membranes of bovine retina and pigment epithelium and by altering their stability causes release of lysosomal enzymes. The observation that the proteolitic and glucuronidase activities are released in the presence of retinol in percentage greater than that obtained after ultrasonic treatment, may be attributed to some enzyme activation process. Previous research has brought to light numerous conditions, both experimental and pathologic, in which an accumulation of retinol, or a series of conditions which might lead to it, takes place and is often accompanied by lysosomal damage. The amount of retinol measured in the pigment epithelium of young (24 weeks) rats affected by retinal dystrophy is at least double that at present in the pigment epithelium of normal rats (Reading, 1966). Amemiya (1967) has shown that, in animals in conditions of hypervitaminosis A, there are conspicuous changes in the outer segments and electron microscopic examination showed that the first changes take place in the pigment epithelium.
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In rats affected by retinal dystrophy, the rod outer segments are altered between the 12th and 14th days of age (Dowling, 1962) and the phagocytosis of the pigment epithelium of these animals are altered from the 15th day of age until it disappears (Herron, 1970). At the same age-period the lysosomes of the pigment epithelium of dystrophic rats show serious damage to their wall accompanied by the release of acid phosphatases, a damage which seems to precede retinal dystrophy (Yates, 1970). Our finding that retinol causes release and in some cases even activation of lysosomal enzymes in the retina and pigment epithelium in proportion to its concentration, has led us to assume that, in vivo, an accumulation of retinol might lead to similar result. If this would be the case in vivo, the inevitable consequence of the retinol effect on the release of lysosomal enzymes would be gradual retinal damage. Furthermore the normal lysosomal activity of the pigment epithelium, indispensable for the normal phagocytic activity of these cells, would also suffer. REFERENCES Amemiya, T. (1967). A& Sot. Ophtkal. Yap. 71, 2236. Anson, M. L. (1938). J. Gen. Physiol. 22, 79. De Duve, C., Wattiaux, R. and Wibo, M. (1962). Biochem. Pharmacol. 9, 97. Dingle, J. T. (1961). Biochem. J. 79, 509. Dingle, J. T. (1963). Ciba Foundation Symposium on Lysosomes, p. 384. Ed. by de Reuck, A. V. S. and Cameron, M. P. (Little, Brown and Co., Boston, Mass.). Dingle, J. T., Sharman, I. M. and Moore, T. (1963). Proc. Nut&. Sot. 22, X. Dowling, J. E. and Sidman, R. L. (1962). J. CeZ2 Biol. 14, 73. Fell, H. B. and Mellanby, E. (1953). J. Physiol. (London) 119,470. Fell, H. B., Dingle, J. T. and Webb, M. (1962). Biochem. J. 83,63. Herron, Warren, L., Byron W. Riegel, ORLOE., Myers and Melvin, L. Rubin. (1969). Invest. Opht?dnol. 8, 595. Lucy, J. A. and Dingle, J. T. (1964). Nature (London) 204, 156. Mellanby, E. (1934). J. Pa&Z. Bact. 38,391. Mellanby, E. (1937). Chem. Id (London) 56, 1054. Mellanby, E. (1938). J. Physiol. (London) 93,42. Mellanby, E. (1941). J. Physiol. (London) 99,467. Reading, H. W. (1966). B&hem. J. 100,34p. Thomas, L., McLuskey, R. T., Potter, J. L. and Weissman, G. (1960). J. Exp. Med. 111, 705. Weissman, G. and Thomas, L. (1963). J. Clin. Invest. 42, 661. Weissman, CT., Uhr, J. W. and Thomas, L. (1963). Proc. Sot. Exp. Biol. N. Y. 112, 284. Yates, M. C., Reading, H. W. Bitensky, L. and Chayen, J. (1970). Biochem. J. 119,57 p.