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The metabolism of the dystrophic retina—II
Vision
Ru.
Vol. 4.
pp.
209-220.
Pergamon Press1964.
THE METABOLISM AMINO IN THE
ACID
Printedin GreatBritain.
OF THE DYSTROPHIC
TRANSPORT
DEVELOPING
AND
RAT RETINA,
H. W.
READING
and
PROTEIN
NORMAL ARNOLD
RETINA-II
SYNTHESIS AND
DYSTROPHIC
SORSBY
Wemher Research Unit on Ophthalmological Genetics (Medical Research Council), Royal College of Surgeons of England, London (Received 19 November 1963) Abstract-Protein synthesis in the rat retina as determined by the rate of incorporation of Cl” labelled glycine is significantly reduced in the developing dystrophic retina compared with the developing normal retina. This anomalous pattern is seen at 6-8 days after birth; i.e. some 6-8 days before there is histological evidence of degeneration. These findings contrast sharply, on a time basis, with the known anomalies in glucose catabolism which occur only after histological changes are well established. IN A PREVIOUS
study READING and SORSBY (1962) discussed the history of a congenital abnormality in rats, designated as retinal dystrophy. In this affection there is a failure in the final stages of development in the rod layer of the retina. The rods, instead of going on to maturity, degenerate with subsequent degenerative changes in other layers. Biochemically, they found no striking differences in the overall pattern of aerobic glucose metabolism between normal and dystrophic rat retinae during the period of 10-21 days after birth; i.e. the period when pathological changes develop in the affected animal. These investigations covered glucose catabolism by the glycolytic and direct oxidative (Krebs’s cycle) pathways. However, it was found that with increase in age over this period, the dystrophic retina, unlike its normal counterpart, failed to retain intracellularly some of the amino acids which the tissue forms directly from glucose. The “leakage” of amino acids from the tissue in vitro suggests a breakdown in the proper utilization of energy required for transport mechanisms, an effect which could also be manifest as a reduction in the rate of biosynthetic reactions within the cell. Whilst either of these possibilities might lead to diminished rate of protein synthesis, this could also be affected if the intracellularly formed amino acids are present in inadequate quantities in the dystrophic retina. With these possibilities in mind the present investigation, comprising a study of the uptake, transport and incorporation into retinal protein, of radioactively labelled glycine both in vitro and in viva was carried out. EXPERIMENTAL
PROCEDURES
Animals and Preparation of Tissues Pink-eyed piebald agouti rats, originally from the stock described by BOURNE et al. (1938), affected with the retinal dystrophy, were used throughout, and for comparison normal, black-hooded (PVG) rats bred from stock originally obtained from Glaxo Laboratories Ltd. All experiments were carried out on litter-mates irrespective of sex. The preparation of retinae for biochemical study has been described previously (READING and SORSBY, 1962). 209
0
Incubation Conditions jar in vitro E.vpcritmwts Glycine uniformly labelled Cl1 (obtained from the Radiochemical Centre, Amersham) with a specific activity of 2.19 mc,‘mM was added to the medium bathing the tissue in in 1-O ml amounts which brought the activity in each flask to 5.0 ,DC(2.39 PM). Incubations (total volume) medium were carried out in 5-ml micro-Warburg flasks attached to manometers in an atmosphere of pure oxygen, using about 20-30 mg (wet weight) of retinal tissue per flask. Non-radioactive glucose was added to each flask in a final concentration of 0.1 e{,. The inorganic salt medium used was Krebs’s original Ringer-phosphate (DAWSON et al., 1959). This was chosen in view of the report by BASSI and BERNELLI-ZAZZERA (1960) that incubation of brain slices in a potassium-rich phosphate medium increased respiration, but at the same time markedly inhibited amino acid incorporation into protein. Carbon dioxide was absorbed on filter-paper rolls soaked in 30% KOH placed in the centre-wells of the Warburg flasks. All experiments were carried out under identical conditions; viz. the inorganic salt medium together with glucose was placed in the main compartment of each flask whilst radioactive glycine solution was placed in the side-arm. Immediately after dissection, the retinal tissue was added to the contents of the main compartment of each previously chilled flask. The flasks were then attached to manometers and after gassing and temperature equilibration the radioactive glycine in the side-arm was added to the tissue, etc., in the main compartment. Incubation was continued for 2 hr at 37”C, oxygen uptake being measured at 15 min intervals. After incubation, the flasks and contents were rapidly chilled prior to the fractionation procedure. Fractionation Procedure The fractionation procedure described below was similar in outline to that described by SIEKEVITZ (1952) with certain modifications. After centrifugation to separate retinal tissue and medium, the tissue was washed twice in physiological saline, then homogenized in cold 10% trichloracetic acid. The trichloracetic acid extracts were frozen and kept for assay. Residues were treated at 90°C for 20 min in 5 % trichloracetic acid to remove nucleic acids. Lipid was then removed by extracting the residues consecutively with (a) 95% ethanol, Final traces of solvent were (b) ethanol: ether : chloroform, 2 : 2 : 1 mixture, and (c) ether. removed under reduced pressure. Measurement cf RudioactivitJ1 A number of methods were tried for plating out the protein residues in a state suitable for accurate counting. One of the major difficulties encountered was in the small quantity of protein available, but finally a procedure similar to that described by ROODYN et al. (1962) was adopted. Protein residues were dissolved in 0.2-O-4 ml 98% formic acid and plated on 3*0 cm2 aluminium planchets, the surface of which was covered with a circle of lens tissue attached by means of silicone grease. By this means an evenly distributed protein film without “curling” was obtained. Planchets were dried overnight over KOH and the amount of protein on each one determined by weighing. Counts were made under a thin end-window Geiger counter, and corrected to infinite thinness from a calibration curve prepared by plating differing amounts of a standard preparation of total liver protein of known specific C 14 Perspex source obtained from the radioactivity, in comparison with a standard Radiochemical Centre. Evaluation of Cl4 glycine incorporation v,‘as based on determinations of specific activity of the respective protein samples.
The Metabolism of the Dystrophic Retina-II
211
Aliquots of the incubation medium and trichloracetic acid extracts of retinal tissue were subjected to paper chromatography using a bi-dimensional solvent system originally described by CHAIN et al. (1960). The position of free radioactive glycine on the chromatograms was determined by making autoradiographs on X-ray film. Characterization was effected by adding non-radioactive glycine to the extract spot at the source, and after running the chromatogram and spraying with ninhydrin the position of the glycine “spot” was found to correspond to the area of radioactivity on the autoradiograph. Quantitative evaluation of the radioactivity of Cl4 glycine on the papers was carried out in an automatic scanning device (FRANK et al., 1959). In addition, where possible, aliquots of medium and extract were subjected to electrolytic de-salting, then plated out on to 3.0 cm2 aluminium planchets (with lens tissue inserts), dried in vacua over KOH and counted at infinite thinness under a thin end-window Geiger counter. Good correlation between the two counting techniques was obtained. In vivo Experiments In this series of experiments, 8-day-old litter mates of either the PVG (normal) or Campbell (dystrophic) strain were injected subcutaneously with a saline solution of uniformly Cl4 labelled glycine, 10 PC per animal. Animals were killed in pairs at intervals of 1, 3, 6 and 24 hr after injection. After killing the animals by cervical fracture, the eyes were removed and the retinae dissected out as rapidly as possible, washed in ice-cold physiological saline, the excess moisture removed, and then weighed on a torsion balance. The four retinae from each pair of rats were pooled and homogenized in 2 x 1-Oml quantities of 10 % trichloracetic acid. Subsequent fractionation, plating out and counting of retina1 protein was carried out exactly as described for the in vitro experiments. Livers were also removed from each animal, washed in saline, blotted dry, weighed, homogenized in 2 x 5 ml quantities of 10% trichloracetic acid, then treated as for retina. In order to maintain comparative experimental conditions, the dried liver protein powder was well mixed, and small aliquots (3-4 mg) used for radioactivity measurements. Trichloracetic acid extracts of retinal and liver tissue were electrolytically de-salted, plated out, dried in vacua over KOH and assayed for radioactivity under a thin end-window Geiger counter. Aliquots were chromatographed to ascertain that the radioactivity arose from free glycine. Check for adsorbed radioactivity andprotein content of precipitates. It is always possible that some or even all of the radioactivity associated with the protein fractions obtained by the procedures outlined above could be due to adsorbed radioactive amino acid and therefore not represent true amino acid incorporation. In order to eliminate this possibility in the present experiments a number of representative samples of the dried retinal protein obtained after incubation of the tissue with Cl4 glycine were taken and subjected to degradation by the ninhydrin-carbon dioxide method of VAN SLYKE et al. (1941). By this means, any free amino acid, as opposed to amino acid incorporated, is decarboxylated at the a-carboxyl position and the carbon dioxide formed is trapped as barium carbonate. If in the free amino acid the carbon at the a-carboxyl position is labelled C14, then it follows that the barium carbonate is also Cl4 labelled and can be assayed. Amino acid incorporated into the protein molecule does not undergo any reaction in this procedure. In all the samples examined, adsorption was found to account for less than 0.1 per cent of the amount of glycine actually incorporated.
H. W.
212
READING
AND
ARNOLD
SORSB~
The nitrogen content of representative samples of retinal protein precipitates was checked by the micro-KjeldhaI method to examine the possibility of accidental inclusion of non-protein material. RESULTS
In vitro Experiments Oxygen uprake. The rates of oxygen uptake were measured to obtain some idea of the viability of the tissue samples used. Figure 1 shows the mean values for the oxygen uptake over the 2-hr incubation period.
A80 70
60
50
-I
40
30
t
1
I
i
I
I
I
6
8
IO
I2
i6
24
DAYS
Cf= AGE
uptake of normal and dystrophicrat retinae in Gtro. Ordinates-p1 oxygen per 25 mg (wet wt.) tissue/2 hr.
FIG. 1. Oxygen
‘C)Normal
0 Dystrophic
Oxygen uptake in the dystrophic retina at 8-12 days was higher than in the unaffected tissue. but thereafter a gradual fall-off occurred, whereas uptake in the normal retina continued to rise steadily up to 24 days. These comparative curves represent total uptake over a 2-hr period with a bicarbonate-containing incubation medium and show a slightly different pattern from those obtained previously with glucose as the soIe carbon substrate and a phosphate medium. The significance of the early stimulation of oxygen uptake in the dystrophic retina, which is not maintained, is not clear, uniess it is an indication that under the incubation conditions employed there is an uncoupling of oxidation and phosphorylation in the dystrophic retina. Uptake of Cl4 glycine. Table 1 shows the uptake of Cl4 glycine from the incubation medium. Rates of uptake in both normal and affected rat retinae appear to be fairly constant, during the period of differentiation and development of the lesion, since none of the differences at particular ages was significant (lowest value for P was O-2). Free amino acid concentration in the tissue. Since radioactive glycine taken up from the external medium into the retinal cells enters into the amino acid pool before incorporation, at equilibrium the level of radioactivity in tissue extracts is a measure of the rate of transport of glycine. Table 2 presents the values for the concentration of free Cl4 glycine found
213
The Metabolism of the Dystrophic Retina-II
TABLE 1. UPTAKEOF U-Cl4 GLYCINE FR~~MINCUBA~ION MEDIUM Values in 10"x FM gtycine/mg (wet wt) tissue/Z hr. Standard errors of the means in parentheses.
Each value represents the average of six to nine experiments.
Dystrophic
Age b-kws)
Normal
6 8 10 I2
166 (13) 21.5 (44) 240 (40) 231 (91) 222 (77) 240 (40)
;:
205 (26) 277 (30) 236 (24)
Significance of difference between means PLO.2 P -’ 0.3 P --04 P -- 0.6 P -- 0.6 P 0.8
160 155)
168 (34) 219 (56)
in trichloracetic acid extracts of retina. In all except one case, no statistically significant differences were observed between normal and affected animals. Twenty-four-day-old dystrophic retinae showed a significantly lower (P=O*O2) tissue concentration of Cl1 glycine than corresponding normal retinae. TABLE 2. CONCENTRATION OF FREE U-Cl4 CLYCINE IN EXTRACTS OF RETINAL TISSUE i!rvitro Values in 10” x ,uM glycine/mg (wet wtf tissue. Standard errors of the means in parentheses. Each value represents the average of six to nine experiments.
Age (days)
Normal
Dystrophic
Significance of difference between means
6 8
30 (2) 27 (12) 28 (16)
29 (3) 22 (8) 2t (4)
P-O.8 P=O*7
:(:
21 (6)
24 (3
P-O.6
;:
28 (10) 37 (5)
32 t4 (4) (2)
P-O.6 p ==0.02
pz.o.7
Protein synthesis. Table 3 presents the results of the measurement of ClJ glycine incorporation into total retinal protein, based on the relative specific activities of the protein samples. Statistically significant differences (P=O+M) were found between incorporation rate into protein of normal retinae and into retinae taken from dystrophic animals, at ages 6 and 8 days. At 10 days of age, the difference between the mean incorporation rates was of low significance (P=O*O7) and at later ages, viz. 12-24 days, the differences were not TABLE 3. INCORPORATION
OF U-Cl”
GLYCINE INTO RETINAL PROTEINS
vitro
Values in 10” x yM giycine per mg. dried protein. Standard errors of the means in parentheses. represents the average of approximately ten experiments.
Age (days)
Normal
8
167 (t5) 148 (18)
10
81 (5)
I2 16 24
78 (7) 42 (9) 14 tt1
6
Dystrophic 118 (16) 100 (6) 59 (10) 60 (7) 3t (5) 17 (3)
Each value
Significance of difference between means P-O@4 P
Pt0.07 PzO.10 P-O.30 P --0.40
214
H. W. REAIIING AND ARNOLD SORSB~
significant. However, the series of results taken as a whole showed a definite trend towards an equal rate of incorporation in both normal and affected animals coincident with the retina reaching full development. In vivo Experiments
on 8-day-old
Rats
Transport of Cl4 glycine into retina and incorporation itlto retinul protein. Tables 4 and 5 show the concentration of Cl4 glycine found in trichloracetic acid extracts of retinal and liver tissue respectively at the stated intervals after injection of Cl” glycine. As in the in vitro experiments these figures represent a measure of the transport of Cl4 glycine into the tissue. Comparison of normal and dystrophic animals showed no real differences between either strain in retina or liver. TABLE 4. CONCENTRATION OF FREE U-F-
GLYCINE IN EXTRACTS OF RETINAL TISSUE NI I~J
Values in 104 x pM glycine per 100 mg. (wet wt.) tissue. Each value is the mean of three or four determinations, with limits quoted in parentheses. Time after injection (hr) I 3 6 24
Normal
Dystrophic
65 (i-5)
64 (t3) 32 (13) 14 (t6) IO (&2)
29 (53) 14 (54) 1I (&3)
TABLE 5. CONCENTRATION OF FREE U-C’” Values in lOa xpM
GLYCINE IN EXTRACTS OF LIVER TISSUt i/l\'iWJ
glycine/lOO mg (wet wt.) tissue. Each value is the mean of
Time after injection (hr) 1 3 6 24
Normal 108 70 28 21
three determinations.
Dystrophic 138 56 35 28
Figure 2 presents data on the incorporation of Cl” glycine into total retinal protein following injection of radioactive glycine. A small and probably insignificant difference was observed between normal and dystrophic animals 1 hr after injection, whereas 3 hr after injection the higher incorporation in the normal retina was quite marked. Subsequently, at 6 and 24 hr after injection, the incorporation figures were higher inthe dystrophic animals. Figure 3 presents similar comparative incorporation data in the livers of normal and dystrophic rats. No marked differences were apparent between the two strains, incorporation being more rapid in liver (i.e. peak at 1 hr or less) than in retina (peak at 3 hr) in either strain. The decrease in incorporation figures with increasing time after injection, due to loss of labelled amino acid (protein degradation), proceeded at equal rates in the liver in either strain.
The Metabolism
215
of the Dystrophic Retina-11
P
I 1
3
6
24
HOURS
FIG. 2. In vivo comparison of rates of incorporation of Cl4 glycine into retinal protein by normal and dystrophic rats, following subcutaneous injection of the labelled amino acid (10 flc per animal).
Ordinates--Incorporation of Cl4 glycineexpressed as IO5 x pmole glycine/mg protein. Abscissae-hr after injection. G Normal 0 Dystrophic
I
3
6
noms
FIG. 3. 1~ vivo comparison of rates of incorporation of glycine’” into liver protein by normal and dystrophic rats following subcutaneous injection of the labelled amino acid (IO PC per animal). Ordinates-Incorporation of Cr4 glycine expressed as lo” 7 Innok glycinc/mg protein. Abscissae-hr after injection. CI Normal l Dystrophic
24
216
H. W. READING
AND
AKNOLIJ
SORSBI
DISCUSSION
incorporation of Cl3 Glvcine irlto RetinaI Protein in the Normal and the D?strophic Retitta
In vitro~~~~~~~. The most striking results of the present series of experiments are those concerning the incorporation of labelled amino acid into total retinal protein. The high initial rate of amino acid incorporation and its decrease with increase in age in the immature growing retina, and the establishment of a steady “turnover” rate after differentiation, parallels the situation observed in developing mammalian brain as described by RICHTER (1955). The implication is that protein synthesis must keep pace with the rapidly increasing growth of the tissue. The differences between retinae taken from normal rats and those taken from dystrophic rats in vitro were quite marked at 6 and 8 days of age, the dystrophic animals showing a much lower rate of incorporation. Thereafter, in older animals, the trend of the normal retina towards a higher rate of amino acid incorporation persisted but the differences between normal and affected retinae were not so marked, although the decrease in statistical significance with increase in age was gradual. The marked differences of incorporation rate, which is a measure of net protein synthesis, are apparent before any histological damage can be discerned in the retina. In vivofi&ings. Confirmation of the decreased rate of protein synthesis in the dystrophic retinae was obtained from the in vivo experiments. Following injection of Cl” labelled glycine into 8-day-old litter-mate rats, the maximal incorporation rate into total retinal protein was lower in the dystrophic than in the normal rats. On the other hand, the fact that no differences were found in maximal incorporation of labelled amino acid into liver protein between the normal and dystrophic animals is good evidence that the findings for retina are not due simply to differences of strain. Although amino acid incorporation 3 hr after injection is less in the dystrophic retina. there is evidence of a slower rate of protein breakdown and degradation in the affected tissue. This tendency is important since it is always possible that an apparent reduction in incorporation of labelled amino acid could be caused by an increased rate of protein breakdown. This does not appear to be the case in this instance, and the present results are compatible with established ideas on protein metabolism. Conditions which limit the release or utilization of energy depress protein synthesis but also depress its breakdown and so depress the consequent rate of liberation of amino acids (STEINBERGand VAUGHAN, 1956; CAMPBELL,1958). The suggestion is that the two processes of protein synthesis and breakdown are interrelated and that both require phosphate-bond energy sources. The Nature of the Reduced Rate of Protein Synthesis in the Dystrophic Retinn It appears from both the in viro and in vitro experiments that the limitation in rate of net protein synthesis in the dystrophic animal is not due to reduced amino acid transport into the tissue. The influx of free glycine into either normal or dystrophic retina was the same in each instance; both showed similar maximum tissue concentration at the same time after injection (in viva) or during the incubation period (in vitro). Although the rate of protein synthesis was high in very young retinae and decreased with increase in age, the intracellular glycine concentration differed very little over the period examined. This observation is not surprising since the two processes are known to depend on different factors. For instance, certain synthetic amino acids enter transport mechanisms but not protein synthesis, and RIGGS and WALKER (1963) have recently shown in isolated cells that amino acid transport
The M~tab~~lism of the Dystrophic Retina-11
117
and protein synthesis are independent of one another. except as active transport is necessary to supply the amino acids for incorporation. In other words, incorporation can be altered by varying free amino acid level in the cell, but above a certain threshold concentration of amino acid, incorporation is not affected. The same authors also found that amino acid transport appeared to be less sensitive to an adequate supply of energy than did amino acid incorporation into protein. The present results, considered in the light of the above factors, suggest that the lower rate of protein synthesis in the dystrophic retina could be a consequence of an impairment in energy utilization and not a direct result of impairment in the amino acid transport process, the “defect” in energy utilization probably not being marked enough to affect transport during the early stages. It was thought from our previously reported investigation on glucose metabolism that amino acid transport might be directly implicated, since intracellularly formed amino acids tended to “leak” out into the incubation medium from the dystrophic retina. The present experiments confirm this leakage to a certain extent, since at 24 days intracellular glycine concentration was signi~cantly less in the dystrophi~ retina than in the normal; i.e. at a late stage in the development of the lesion. Whatever the primary cause, the anomaly in protein synthesis in the immature dystrophic retina could reflect either an inability of the tissue to manufacture a specific protein moiety or an inadequacy in general protein synthesis. The latter possibility is thought to be unlikely, since histologically the dystrophic retina grows and develops normally, except that at the end of the developmental period rod cell outer segments do not develop fully, and a general degeneration of the entire rod cell layer follows. Most genetically induced abnormal conditions associated with protein metabolism at the cellular level are associated either with an absence or reduction in a particular enzyme activity (e.g. galactosaemia), or alteration in structure or rate of synthesis of a particular non-enzymic but physiologically functional protein (e.g. various anaemic conditions involving one or other of the haemoglobin chains), or else a gross deficiency in synthesis of a specific protein (e.g. analbuminaemia). An excellent review of present knowledge in this field has recently been given by HARRIS (1963). It appears unlikely that the genetically controlled abnormality presented by the retinal dystrophy is concerned with deficiency of a particular enzyme. Enzyme proteins occur in only very small quantities, so that a difference in incorporation rate, as observed here, would probably not be so marked if dealing with a simple enzyme deficiency. It is more likely that a reduced rate of synthesis of a non-enzymi~ but nevertheless functionally important protein is involved in the retinal dystrophy, which is normally present in the tissue in moderate quantity. R~~at~~n~h~of Observed Changes to the Protein Moiety of Visual Purple Such considerations applied to retina cannot but raise the question of the involvenlent of opsin, the protein moiety of the visual pigment. It fulfils the requirement of being produced in quantity, plays a part in the visual cycle and is structurally localized in the lamellar structure of the rod outer segments (DE ROBERTS, 1960), the structures which show the first signs of degeneration in the rat dystrophy. However, there are certain pieces of conflicting evidence which must be examined before the question of the involvement of opsin can be decided positively or negatively. BONTING et al. (1961), in a recent study of the developmental aspects of the rhodopsin cycle in the albino rat, did not detect rhodopsin formation until 6 days after birth, and appreciable quantities were not found until after 10 days. It would appear that protein synthesis as measured by amino acid incorporation rate is
inversely propo~ional to the rate of rhodopsin formation. The spectroscopic estimation of rhodopsin depends, ofcourse, on the optical absorption characteristics of the entire molecule, and especially that of the chromophoric (retinene) group. It is possible that opsin itself may be present in the immature rod cells before organized synthesis of the visual pigment itself occurs. Concerning the nature of rhodopsin in the dystrophic rat. DOWLING and SIDMAN (1962) have shown that the visual pigment from affected retinae has an entirely normal spectrum, and presumed from this that the structure is normal. This conclusion makes the necessary assumption that changes in opsin structure would alter the complete optical characteristics of rhodopsin found in the affected retina. This assumption may or may not be correct, since such changes in opsin structure would be likely to concern the make-up of peptide chains in the molecule only. The same authors aiso found that the dystrophic animals had considerably more rhodopsin than corresponding normal controls up to about 30 days of age, after which the rhodopsin concentration in the affected eyes fell rapidly. These authors concluded that the loss of rhodopsin after 30 days of age reflected an inability to resynthesize rhodopsin after bleaching. At first sight the present findings of an early reduction in protein synthesis and Dowling and Sidman’s demonstration of over-production of rhodopsin in dystrophic rats seem irreconcilable, but it should be remembered that here we are measuring the rate of incorporation of amino acid into protein, a measure of net protein synthesis, not a measure of the amount of total retinal protein. It can be seen from the results of the in ri~o experiments that both a depression in rate of protein synthesis and breakdown (i.e. turnover rate) occurs in the dystrophic retina; if a rapid turnover is normal (which it appears to be) then a decrease in this rate is likely to be reflected in an increase in amount of total protein present at any particular instant. It is suggested that the anomaly in protein synthesis in the dystrophic retina results in a reduction in opsin turnover, The increase in total rhodopsin content, as described by Dowiing and Sidman, is possibly a manifestation of a feedback mechanism in an attempt by the affected tissue to conserve rhodopsin. The dramatic drop in rhodopsin content at about 30 days could be the point of breakdown of such a feedback mechanism. Such an imbalance in the visual cycle in the dystrophic retina is likely to be reflected in changes in dynamically linked mechanisms. Some recent work by FWTTERMAN(1963) may be pertinent to this problem. Futterman demonstrated that mammalian visual cell outer segment preparations can metabolize glucose by the “pentose shunt” pathway as well as by glycolysis, and that the “shunt” is implicated in the visual cycle, inasmuch as the reduced nucleotide co-enzyme NADPHs produced during its operation is the principal agent for the reduction of retinene to vitamin A alcohol following light stimulation. It seems plausibIe, therefore, that alteration in the balance of the visual cycle may be reflected by changes in the NADPHsiNADP ratio with concomitant changes in the operation of the shunt mechanism. Whether such changes occur early or late in the course of the development of the dystrophy remains to be seen. SUMMARY By using Cl” labelled glycine. the rate of protein synthesis and the transport of amino acid have been studied both in vitro and in viva in the retina of normal rats and of rats affected with retinal dystrophy, an inherited condition resembling human retinitis pigmentosa. In v&o studies were carried out on retinae from rats over the age range of 6 to 24 days; in vivo studies were confined to rats aged 8 days. The following results were obtained.
The Metabolism of the Dystrophic Retina-II
219
1. In tpitro dystrophic retinae showed a significantly lower rate of incorporation of glycine into total protein than normal retinae at 6 and 8 days of age. At 10 to 24 days the differences, though present, were less marked, until by 24 days incorporation rates were equal. 3 No differences in amino acid uptake from the incubation medium, nor in transport of -. amino acid into the retinal tissue, between normal and affected retinae. were detected in the early stages, but at 24 days the intracellular concentration of glycine was markedly lowered in dystrophic retinae. 3. These in vitro results were confirmed by measuring the incorporation of Cl1 glycine into retinal protein at various intervals following the subcutaneous injection of the labelled amino acid into live intact 8-day-old litter-mate rats. C.ompared with unaffected rats the dystrophic animals showed a marked diminution in incorporation rate (protein synthesis) and, in addition, a slower rate of protein breakdown. Since amino acid incorporation rates into total protein in the livers of normal and affected rats were equal, it may be taken that the differences observed in the retina were not simply due to differences in strain of animal. As in the in vitro experiments, amino acid transport into the retina was equal in normal and dystrophic animals. 4. In an earlier study we have shown that certain changes in the carbohydrate metabolism of the retina are observed only subsequent to the histological degenerative changes in the rods. The anomalous protein synthesis recorded here occurs some 6-8 days before histological changes can be seen in the dystrophic retina-in fact before the retina has become fully differentiated into its various layers. Ackno,u/edge,llen?s-We
are greatly indebted for loyal technical assistance to Mrs. DIANA B~LT~N and secretarial help.
Mr. IVAN P. GREEN and to Miss E. M. GOWER for painstaking
REFERENCES BASSI, M. and BERNELLI-ZAZZERA, A. (1960). Effects of potassium ions in brain respiration and amino acid incorporation into brain proteins in vitro. Experientia 16, 430. BONTING, S. L., CARRAVAGGIO, L. L. and GOURAS, P. (1961). The rhodopsin cycle in the developing vertebrate retina-l. Relation of rhodopsin content, electroretinogram and rod structure in the rat. Exp. E>v Res. 1, 14-24. BOURNE, M. C.. CAMPBELL, D. A. and TANSLEY, K. (1938). Hereditary dcgcneration of the rat retina. Brit. J. Ophtltal.
22, 613-623.
CAMPBELL, P. N. (1958). Protein synthesis with special reference to growth processes both normal and abnormal. Advances in Cancer Research, V, pp. 98-155. Academic Press, New York. CHAIN, E. B., LARSSON, S. and POCCHIARI, F. (1960). The fate of glucose in different parts of the rabbit brain. Proc. ray. Sot. B152, 283-289. DAWSON, R. M. C., ELLIOT, D. C., ELLIOT, W. H. and JONES,K. M. (1959). Data,for Biorhrrnica/ Research. Clarendon Press, Oxford. DE ROBERTIS, E. (1960). Some observations on the ultrastructure and morphogcnesis of photoreceptors. J. gen. P/rysio/. 43, l-l 3. DOWLING, J. E. and SIDMAN, R. L. (1962). Inherited retinal dystrophy in the rat. J. cc//. Biol. 14, 73-IOV. FRANK, M., CHAIN, E. B.. POCCHIARI, F. and Rossr, C. (1959). A modified apparatus for the quantitative evaluation of radio-paper-chromatograms. Selected Scientific Papers. 131.Slrp. Smiro 2, 75-87. FUTTERMAN.S. (1963). Metabolism of the retina. 111.The rble of reduced triphosphopyridinc nuclcotidc in the visual cycle. J. biol. Chem. 238, 1145-l 150. HARRIS, H. (1963). Garrod’s Inborn Errors ofMetabo/isw, pp. 120-197. Oxford University Press, London. READING. H. W. and SORSBY, A. (1962). The metabolism of the dystrophic retina. I. Comparative studies on the glucose metabolism of the developing rat retina, normal and dystrophic. Vision Rcs. 2. 315-325. RICHTER, D. ( 1955). In Bioclwr~istry of’ fhe dewlophg nenms .swetv, pp. 241-243. Ed. WAELSCH, H. Academic Press. New York. RIGGS, T. R. and WALKER, L. M. (1963). Some relations between active transport of free amino acids into cells and their incorporation into protein. J. biol. C/rem. 238, 2663-2668.
220
H. M’.
READING
ANII
ARWLII
SORSB~
RO~DYN, D. B., SUTTIE, J. W. and WORK, T. S. (1962). Protein synthesis in mitochondria. Blorlrcr~~. J. 83, 2940. SIEKEVITZ, P. (1952). Uptake of radioactive alanine i/t Gtro into the proteins of rat liver fractions. d. hiol. Chew. 195, 549-565. STEINBERG. D. and VAUGHAN, M. (1956). Observations in intracellular protein cat‘~bolism studied ;/I I,;/IYI.
Arch.
Biochem.
Biophys.
65, 93-105.
VAN SLYKE, D. D., MACFADYEN, D. A. and HAMILTON. P. (1941). by titration of the carbon of the carbon dioxide formed in the
The determination
of fret amino acids
reaction with ninhydrin.
J. hiol. C/WW.
141, 671.
R&urn&-La synthese de protkine dans la retine du rat, determini par la vitesse d’incorporation de glycine marquee B Cl” est notamment plus reduit dans le cas de la rktine dystrophique en tours de dCveloppement que dans la rttine normale dCveloppante. Cet anomalie devient apparente 6-8 jours aprts la naissance; c’est g dire 6-8 jours avant I’&idence histologique de d&&ration. Ces decouvertes ne sont nullement d’accord, du point de vue du temps, avec les anomalieb connues de la catabolisme de glucose, qui ne se produisent qu’une fois les changements histologiques sont bien ttablies.
Zusammenfassung-Die
Proteinsynthese in der Netzhaut von Ratten wie sie durch den Einbau von mit Cl4 markiertem Glycin gemessen wird, ist in der sich entwickelnden dystrophischen Netzhaut deutlich geringer als in der sich entwickelnden normalen Retina. Dieses anormale Verhalten wird 6-8 Tage nach der Geburt beobachtet, also etwa 6-8 Tage bevor sich der histologiche Befund der Degenerierung zeigt. Die Ergebnisse stehen beziiglich der zeitlichen Reihenfolge im Gegensatz zu den bekannten Anomalien im Glukose Katabolismus, die erst auftreten, wenn die histologischen Verinderungen schon gut ausgebildet
sind.
PeNOM&--CUHTe3 6eJlKa B PeTHHe KpblCbl, OllpeNl~eMblti Ha OCHOBaHUU CKOPOCTII BKJlKNeHHII rJlUUUHa MeYeHOI-0 cl4 3Ha’fHTJlbHO YMeHblUaeTCfi B XOJle pa3BUTUN ,UHCTpO&iYeCKW U3MeHeHHOZt FTUHbl lI0 CpaBHeHUlO C pa3BUBalOLUefiC~ HOpMaJIbHOfi PeTUHOti. 3Ta aHOManbHaR Kaj?THHa BHLIHa yme Ha 6-8 IleHb IlOCJle pO)KHcneHUn, T.e. 38 6-8 AHe&? LIO TOrO, KPK AW2HepZiWiR MOIKeT 6blTb 06HapynCeHa I-UCTOJlOrUqeCKU. qT0 pe3KO OTIlA’laeTCII HO BpeMeHHblM llOKa3aTennM OT U3BeCTHbIX aHOManUfi B MeTa6onu3Me rJlIOK03bl llpU AUCCUMUllRUHU rJlK)K03bl, KOTOpble 06HapyIKUBatOTCR TOJIbKO nocne Tore, KaK rucTonoruqecKWe n3MeHeHuR yxe xopouro BbipaneUbl.
Ru.
Vol. 4.
pp.
209-220.
Pergamon Press1964.
THE METABOLISM AMINO IN THE
ACID
Printedin GreatBritain.
OF THE DYSTROPHIC
TRANSPORT
DEVELOPING
AND
RAT RETINA,
H. W.
READING
and
PROTEIN
NORMAL ARNOLD
RETINA-II
SYNTHESIS AND
DYSTROPHIC
SORSBY
Wemher Research Unit on Ophthalmological Genetics (Medical Research Council), Royal College of Surgeons of England, London (Received 19 November 1963) Abstract-Protein synthesis in the rat retina as determined by the rate of incorporation of Cl” labelled glycine is significantly reduced in the developing dystrophic retina compared with the developing normal retina. This anomalous pattern is seen at 6-8 days after birth; i.e. some 6-8 days before there is histological evidence of degeneration. These findings contrast sharply, on a time basis, with the known anomalies in glucose catabolism which occur only after histological changes are well established. IN A PREVIOUS
study READING and SORSBY (1962) discussed the history of a congenital abnormality in rats, designated as retinal dystrophy. In this affection there is a failure in the final stages of development in the rod layer of the retina. The rods, instead of going on to maturity, degenerate with subsequent degenerative changes in other layers. Biochemically, they found no striking differences in the overall pattern of aerobic glucose metabolism between normal and dystrophic rat retinae during the period of 10-21 days after birth; i.e. the period when pathological changes develop in the affected animal. These investigations covered glucose catabolism by the glycolytic and direct oxidative (Krebs’s cycle) pathways. However, it was found that with increase in age over this period, the dystrophic retina, unlike its normal counterpart, failed to retain intracellularly some of the amino acids which the tissue forms directly from glucose. The “leakage” of amino acids from the tissue in vitro suggests a breakdown in the proper utilization of energy required for transport mechanisms, an effect which could also be manifest as a reduction in the rate of biosynthetic reactions within the cell. Whilst either of these possibilities might lead to diminished rate of protein synthesis, this could also be affected if the intracellularly formed amino acids are present in inadequate quantities in the dystrophic retina. With these possibilities in mind the present investigation, comprising a study of the uptake, transport and incorporation into retinal protein, of radioactively labelled glycine both in vitro and in viva was carried out. EXPERIMENTAL
PROCEDURES
Animals and Preparation of Tissues Pink-eyed piebald agouti rats, originally from the stock described by BOURNE et al. (1938), affected with the retinal dystrophy, were used throughout, and for comparison normal, black-hooded (PVG) rats bred from stock originally obtained from Glaxo Laboratories Ltd. All experiments were carried out on litter-mates irrespective of sex. The preparation of retinae for biochemical study has been described previously (READING and SORSBY, 1962). 209
0
Incubation Conditions jar in vitro E.vpcritmwts Glycine uniformly labelled Cl1 (obtained from the Radiochemical Centre, Amersham) with a specific activity of 2.19 mc,‘mM was added to the medium bathing the tissue in in 1-O ml amounts which brought the activity in each flask to 5.0 ,DC(2.39 PM). Incubations (total volume) medium were carried out in 5-ml micro-Warburg flasks attached to manometers in an atmosphere of pure oxygen, using about 20-30 mg (wet weight) of retinal tissue per flask. Non-radioactive glucose was added to each flask in a final concentration of 0.1 e{,. The inorganic salt medium used was Krebs’s original Ringer-phosphate (DAWSON et al., 1959). This was chosen in view of the report by BASSI and BERNELLI-ZAZZERA (1960) that incubation of brain slices in a potassium-rich phosphate medium increased respiration, but at the same time markedly inhibited amino acid incorporation into protein. Carbon dioxide was absorbed on filter-paper rolls soaked in 30% KOH placed in the centre-wells of the Warburg flasks. All experiments were carried out under identical conditions; viz. the inorganic salt medium together with glucose was placed in the main compartment of each flask whilst radioactive glycine solution was placed in the side-arm. Immediately after dissection, the retinal tissue was added to the contents of the main compartment of each previously chilled flask. The flasks were then attached to manometers and after gassing and temperature equilibration the radioactive glycine in the side-arm was added to the tissue, etc., in the main compartment. Incubation was continued for 2 hr at 37”C, oxygen uptake being measured at 15 min intervals. After incubation, the flasks and contents were rapidly chilled prior to the fractionation procedure. Fractionation Procedure The fractionation procedure described below was similar in outline to that described by SIEKEVITZ (1952) with certain modifications. After centrifugation to separate retinal tissue and medium, the tissue was washed twice in physiological saline, then homogenized in cold 10% trichloracetic acid. The trichloracetic acid extracts were frozen and kept for assay. Residues were treated at 90°C for 20 min in 5 % trichloracetic acid to remove nucleic acids. Lipid was then removed by extracting the residues consecutively with (a) 95% ethanol, Final traces of solvent were (b) ethanol: ether : chloroform, 2 : 2 : 1 mixture, and (c) ether. removed under reduced pressure. Measurement cf RudioactivitJ1 A number of methods were tried for plating out the protein residues in a state suitable for accurate counting. One of the major difficulties encountered was in the small quantity of protein available, but finally a procedure similar to that described by ROODYN et al. (1962) was adopted. Protein residues were dissolved in 0.2-O-4 ml 98% formic acid and plated on 3*0 cm2 aluminium planchets, the surface of which was covered with a circle of lens tissue attached by means of silicone grease. By this means an evenly distributed protein film without “curling” was obtained. Planchets were dried overnight over KOH and the amount of protein on each one determined by weighing. Counts were made under a thin end-window Geiger counter, and corrected to infinite thinness from a calibration curve prepared by plating differing amounts of a standard preparation of total liver protein of known specific C 14 Perspex source obtained from the radioactivity, in comparison with a standard Radiochemical Centre. Evaluation of Cl4 glycine incorporation v,‘as based on determinations of specific activity of the respective protein samples.
The Metabolism of the Dystrophic Retina-II
211
Aliquots of the incubation medium and trichloracetic acid extracts of retinal tissue were subjected to paper chromatography using a bi-dimensional solvent system originally described by CHAIN et al. (1960). The position of free radioactive glycine on the chromatograms was determined by making autoradiographs on X-ray film. Characterization was effected by adding non-radioactive glycine to the extract spot at the source, and after running the chromatogram and spraying with ninhydrin the position of the glycine “spot” was found to correspond to the area of radioactivity on the autoradiograph. Quantitative evaluation of the radioactivity of Cl4 glycine on the papers was carried out in an automatic scanning device (FRANK et al., 1959). In addition, where possible, aliquots of medium and extract were subjected to electrolytic de-salting, then plated out on to 3.0 cm2 aluminium planchets (with lens tissue inserts), dried in vacua over KOH and counted at infinite thinness under a thin end-window Geiger counter. Good correlation between the two counting techniques was obtained. In vivo Experiments In this series of experiments, 8-day-old litter mates of either the PVG (normal) or Campbell (dystrophic) strain were injected subcutaneously with a saline solution of uniformly Cl4 labelled glycine, 10 PC per animal. Animals were killed in pairs at intervals of 1, 3, 6 and 24 hr after injection. After killing the animals by cervical fracture, the eyes were removed and the retinae dissected out as rapidly as possible, washed in ice-cold physiological saline, the excess moisture removed, and then weighed on a torsion balance. The four retinae from each pair of rats were pooled and homogenized in 2 x 1-Oml quantities of 10 % trichloracetic acid. Subsequent fractionation, plating out and counting of retina1 protein was carried out exactly as described for the in vitro experiments. Livers were also removed from each animal, washed in saline, blotted dry, weighed, homogenized in 2 x 5 ml quantities of 10% trichloracetic acid, then treated as for retina. In order to maintain comparative experimental conditions, the dried liver protein powder was well mixed, and small aliquots (3-4 mg) used for radioactivity measurements. Trichloracetic acid extracts of retinal and liver tissue were electrolytically de-salted, plated out, dried in vacua over KOH and assayed for radioactivity under a thin end-window Geiger counter. Aliquots were chromatographed to ascertain that the radioactivity arose from free glycine. Check for adsorbed radioactivity andprotein content of precipitates. It is always possible that some or even all of the radioactivity associated with the protein fractions obtained by the procedures outlined above could be due to adsorbed radioactive amino acid and therefore not represent true amino acid incorporation. In order to eliminate this possibility in the present experiments a number of representative samples of the dried retinal protein obtained after incubation of the tissue with Cl4 glycine were taken and subjected to degradation by the ninhydrin-carbon dioxide method of VAN SLYKE et al. (1941). By this means, any free amino acid, as opposed to amino acid incorporated, is decarboxylated at the a-carboxyl position and the carbon dioxide formed is trapped as barium carbonate. If in the free amino acid the carbon at the a-carboxyl position is labelled C14, then it follows that the barium carbonate is also Cl4 labelled and can be assayed. Amino acid incorporated into the protein molecule does not undergo any reaction in this procedure. In all the samples examined, adsorption was found to account for less than 0.1 per cent of the amount of glycine actually incorporated.
H. W.
212
READING
AND
ARNOLD
SORSB~
The nitrogen content of representative samples of retinal protein precipitates was checked by the micro-KjeldhaI method to examine the possibility of accidental inclusion of non-protein material. RESULTS
In vitro Experiments Oxygen uprake. The rates of oxygen uptake were measured to obtain some idea of the viability of the tissue samples used. Figure 1 shows the mean values for the oxygen uptake over the 2-hr incubation period.
A80 70
60
50
-I
40
30
t
1
I
i
I
I
I
6
8
IO
I2
i6
24
DAYS
Cf= AGE
uptake of normal and dystrophicrat retinae in Gtro. Ordinates-p1 oxygen per 25 mg (wet wt.) tissue/2 hr.
FIG. 1. Oxygen
‘C)Normal
0 Dystrophic
Oxygen uptake in the dystrophic retina at 8-12 days was higher than in the unaffected tissue. but thereafter a gradual fall-off occurred, whereas uptake in the normal retina continued to rise steadily up to 24 days. These comparative curves represent total uptake over a 2-hr period with a bicarbonate-containing incubation medium and show a slightly different pattern from those obtained previously with glucose as the soIe carbon substrate and a phosphate medium. The significance of the early stimulation of oxygen uptake in the dystrophic retina, which is not maintained, is not clear, uniess it is an indication that under the incubation conditions employed there is an uncoupling of oxidation and phosphorylation in the dystrophic retina. Uptake of Cl4 glycine. Table 1 shows the uptake of Cl4 glycine from the incubation medium. Rates of uptake in both normal and affected rat retinae appear to be fairly constant, during the period of differentiation and development of the lesion, since none of the differences at particular ages was significant (lowest value for P was O-2). Free amino acid concentration in the tissue. Since radioactive glycine taken up from the external medium into the retinal cells enters into the amino acid pool before incorporation, at equilibrium the level of radioactivity in tissue extracts is a measure of the rate of transport of glycine. Table 2 presents the values for the concentration of free Cl4 glycine found
213
The Metabolism of the Dystrophic Retina-II
TABLE 1. UPTAKEOF U-Cl4 GLYCINE FR~~MINCUBA~ION MEDIUM Values in 10"x FM gtycine/mg (wet wt) tissue/Z hr. Standard errors of the means in parentheses.
Each value represents the average of six to nine experiments.
Dystrophic
Age b-kws)
Normal
6 8 10 I2
166 (13) 21.5 (44) 240 (40) 231 (91) 222 (77) 240 (40)
;:
205 (26) 277 (30) 236 (24)
Significance of difference between means PLO.2 P -’ 0.3 P --04 P -- 0.6 P -- 0.6 P 0.8
160 155)
168 (34) 219 (56)
in trichloracetic acid extracts of retina. In all except one case, no statistically significant differences were observed between normal and affected animals. Twenty-four-day-old dystrophic retinae showed a significantly lower (P=O*O2) tissue concentration of Cl1 glycine than corresponding normal retinae. TABLE 2. CONCENTRATION OF FREE U-Cl4 CLYCINE IN EXTRACTS OF RETINAL TISSUE i!rvitro Values in 10” x ,uM glycine/mg (wet wtf tissue. Standard errors of the means in parentheses. Each value represents the average of six to nine experiments.
Age (days)
Normal
Dystrophic
Significance of difference between means
6 8
30 (2) 27 (12) 28 (16)
29 (3) 22 (8) 2t (4)
P-O.8 P=O*7
:(:
21 (6)
24 (3
P-O.6
;:
28 (10) 37 (5)
32 t4 (4) (2)
P-O.6 p ==0.02
pz.o.7
Protein synthesis. Table 3 presents the results of the measurement of ClJ glycine incorporation into total retinal protein, based on the relative specific activities of the protein samples. Statistically significant differences (P=O+M) were found between incorporation rate into protein of normal retinae and into retinae taken from dystrophic animals, at ages 6 and 8 days. At 10 days of age, the difference between the mean incorporation rates was of low significance (P=O*O7) and at later ages, viz. 12-24 days, the differences were not TABLE 3. INCORPORATION
OF U-Cl”
GLYCINE INTO RETINAL PROTEINS
vitro
Values in 10” x yM giycine per mg. dried protein. Standard errors of the means in parentheses. represents the average of approximately ten experiments.
Age (days)
Normal
8
167 (t5) 148 (18)
10
81 (5)
I2 16 24
78 (7) 42 (9) 14 tt1
6
Dystrophic 118 (16) 100 (6) 59 (10) 60 (7) 3t (5) 17 (3)
Each value
Significance of difference between means P-O@4 P
Pt0.07 PzO.10 P-O.30 P --0.40
214
H. W. REAIIING AND ARNOLD SORSB~
significant. However, the series of results taken as a whole showed a definite trend towards an equal rate of incorporation in both normal and affected animals coincident with the retina reaching full development. In vivo Experiments
on 8-day-old
Rats
Transport of Cl4 glycine into retina and incorporation itlto retinul protein. Tables 4 and 5 show the concentration of Cl4 glycine found in trichloracetic acid extracts of retinal and liver tissue respectively at the stated intervals after injection of Cl” glycine. As in the in vitro experiments these figures represent a measure of the transport of Cl4 glycine into the tissue. Comparison of normal and dystrophic animals showed no real differences between either strain in retina or liver. TABLE 4. CONCENTRATION OF FREE U-F-
GLYCINE IN EXTRACTS OF RETINAL TISSUE NI I~J
Values in 104 x pM glycine per 100 mg. (wet wt.) tissue. Each value is the mean of three or four determinations, with limits quoted in parentheses. Time after injection (hr) I 3 6 24
Normal
Dystrophic
65 (i-5)
64 (t3) 32 (13) 14 (t6) IO (&2)
29 (53) 14 (54) 1I (&3)
TABLE 5. CONCENTRATION OF FREE U-C’” Values in lOa xpM
GLYCINE IN EXTRACTS OF LIVER TISSUt i/l\'iWJ
glycine/lOO mg (wet wt.) tissue. Each value is the mean of
Time after injection (hr) 1 3 6 24
Normal 108 70 28 21
three determinations.
Dystrophic 138 56 35 28
Figure 2 presents data on the incorporation of Cl” glycine into total retinal protein following injection of radioactive glycine. A small and probably insignificant difference was observed between normal and dystrophic animals 1 hr after injection, whereas 3 hr after injection the higher incorporation in the normal retina was quite marked. Subsequently, at 6 and 24 hr after injection, the incorporation figures were higher inthe dystrophic animals. Figure 3 presents similar comparative incorporation data in the livers of normal and dystrophic rats. No marked differences were apparent between the two strains, incorporation being more rapid in liver (i.e. peak at 1 hr or less) than in retina (peak at 3 hr) in either strain. The decrease in incorporation figures with increasing time after injection, due to loss of labelled amino acid (protein degradation), proceeded at equal rates in the liver in either strain.
The Metabolism
215
of the Dystrophic Retina-11
P
I 1
3
6
24
HOURS
FIG. 2. In vivo comparison of rates of incorporation of Cl4 glycine into retinal protein by normal and dystrophic rats, following subcutaneous injection of the labelled amino acid (10 flc per animal).
Ordinates--Incorporation of Cl4 glycineexpressed as IO5 x pmole glycine/mg protein. Abscissae-hr after injection. G Normal 0 Dystrophic
I
3
6
noms
FIG. 3. 1~ vivo comparison of rates of incorporation of glycine’” into liver protein by normal and dystrophic rats following subcutaneous injection of the labelled amino acid (IO PC per animal). Ordinates-Incorporation of Cr4 glycine expressed as lo” 7 Innok glycinc/mg protein. Abscissae-hr after injection. CI Normal l Dystrophic
24
216
H. W. READING
AND
AKNOLIJ
SORSBI
DISCUSSION
incorporation of Cl3 Glvcine irlto RetinaI Protein in the Normal and the D?strophic Retitta
In vitro~~~~~~~. The most striking results of the present series of experiments are those concerning the incorporation of labelled amino acid into total retinal protein. The high initial rate of amino acid incorporation and its decrease with increase in age in the immature growing retina, and the establishment of a steady “turnover” rate after differentiation, parallels the situation observed in developing mammalian brain as described by RICHTER (1955). The implication is that protein synthesis must keep pace with the rapidly increasing growth of the tissue. The differences between retinae taken from normal rats and those taken from dystrophic rats in vitro were quite marked at 6 and 8 days of age, the dystrophic animals showing a much lower rate of incorporation. Thereafter, in older animals, the trend of the normal retina towards a higher rate of amino acid incorporation persisted but the differences between normal and affected retinae were not so marked, although the decrease in statistical significance with increase in age was gradual. The marked differences of incorporation rate, which is a measure of net protein synthesis, are apparent before any histological damage can be discerned in the retina. In vivofi&ings. Confirmation of the decreased rate of protein synthesis in the dystrophic retinae was obtained from the in vivo experiments. Following injection of Cl” labelled glycine into 8-day-old litter-mate rats, the maximal incorporation rate into total retinal protein was lower in the dystrophic than in the normal rats. On the other hand, the fact that no differences were found in maximal incorporation of labelled amino acid into liver protein between the normal and dystrophic animals is good evidence that the findings for retina are not due simply to differences of strain. Although amino acid incorporation 3 hr after injection is less in the dystrophic retina. there is evidence of a slower rate of protein breakdown and degradation in the affected tissue. This tendency is important since it is always possible that an apparent reduction in incorporation of labelled amino acid could be caused by an increased rate of protein breakdown. This does not appear to be the case in this instance, and the present results are compatible with established ideas on protein metabolism. Conditions which limit the release or utilization of energy depress protein synthesis but also depress its breakdown and so depress the consequent rate of liberation of amino acids (STEINBERGand VAUGHAN, 1956; CAMPBELL,1958). The suggestion is that the two processes of protein synthesis and breakdown are interrelated and that both require phosphate-bond energy sources. The Nature of the Reduced Rate of Protein Synthesis in the Dystrophic Retinn It appears from both the in viro and in vitro experiments that the limitation in rate of net protein synthesis in the dystrophic animal is not due to reduced amino acid transport into the tissue. The influx of free glycine into either normal or dystrophic retina was the same in each instance; both showed similar maximum tissue concentration at the same time after injection (in viva) or during the incubation period (in vitro). Although the rate of protein synthesis was high in very young retinae and decreased with increase in age, the intracellular glycine concentration differed very little over the period examined. This observation is not surprising since the two processes are known to depend on different factors. For instance, certain synthetic amino acids enter transport mechanisms but not protein synthesis, and RIGGS and WALKER (1963) have recently shown in isolated cells that amino acid transport
The M~tab~~lism of the Dystrophic Retina-11
117
and protein synthesis are independent of one another. except as active transport is necessary to supply the amino acids for incorporation. In other words, incorporation can be altered by varying free amino acid level in the cell, but above a certain threshold concentration of amino acid, incorporation is not affected. The same authors also found that amino acid transport appeared to be less sensitive to an adequate supply of energy than did amino acid incorporation into protein. The present results, considered in the light of the above factors, suggest that the lower rate of protein synthesis in the dystrophic retina could be a consequence of an impairment in energy utilization and not a direct result of impairment in the amino acid transport process, the “defect” in energy utilization probably not being marked enough to affect transport during the early stages. It was thought from our previously reported investigation on glucose metabolism that amino acid transport might be directly implicated, since intracellularly formed amino acids tended to “leak” out into the incubation medium from the dystrophic retina. The present experiments confirm this leakage to a certain extent, since at 24 days intracellular glycine concentration was signi~cantly less in the dystrophi~ retina than in the normal; i.e. at a late stage in the development of the lesion. Whatever the primary cause, the anomaly in protein synthesis in the immature dystrophic retina could reflect either an inability of the tissue to manufacture a specific protein moiety or an inadequacy in general protein synthesis. The latter possibility is thought to be unlikely, since histologically the dystrophic retina grows and develops normally, except that at the end of the developmental period rod cell outer segments do not develop fully, and a general degeneration of the entire rod cell layer follows. Most genetically induced abnormal conditions associated with protein metabolism at the cellular level are associated either with an absence or reduction in a particular enzyme activity (e.g. galactosaemia), or alteration in structure or rate of synthesis of a particular non-enzymic but physiologically functional protein (e.g. various anaemic conditions involving one or other of the haemoglobin chains), or else a gross deficiency in synthesis of a specific protein (e.g. analbuminaemia). An excellent review of present knowledge in this field has recently been given by HARRIS (1963). It appears unlikely that the genetically controlled abnormality presented by the retinal dystrophy is concerned with deficiency of a particular enzyme. Enzyme proteins occur in only very small quantities, so that a difference in incorporation rate, as observed here, would probably not be so marked if dealing with a simple enzyme deficiency. It is more likely that a reduced rate of synthesis of a non-enzymi~ but nevertheless functionally important protein is involved in the retinal dystrophy, which is normally present in the tissue in moderate quantity. R~~at~~n~h~of Observed Changes to the Protein Moiety of Visual Purple Such considerations applied to retina cannot but raise the question of the involvenlent of opsin, the protein moiety of the visual pigment. It fulfils the requirement of being produced in quantity, plays a part in the visual cycle and is structurally localized in the lamellar structure of the rod outer segments (DE ROBERTS, 1960), the structures which show the first signs of degeneration in the rat dystrophy. However, there are certain pieces of conflicting evidence which must be examined before the question of the involvement of opsin can be decided positively or negatively. BONTING et al. (1961), in a recent study of the developmental aspects of the rhodopsin cycle in the albino rat, did not detect rhodopsin formation until 6 days after birth, and appreciable quantities were not found until after 10 days. It would appear that protein synthesis as measured by amino acid incorporation rate is
inversely propo~ional to the rate of rhodopsin formation. The spectroscopic estimation of rhodopsin depends, ofcourse, on the optical absorption characteristics of the entire molecule, and especially that of the chromophoric (retinene) group. It is possible that opsin itself may be present in the immature rod cells before organized synthesis of the visual pigment itself occurs. Concerning the nature of rhodopsin in the dystrophic rat. DOWLING and SIDMAN (1962) have shown that the visual pigment from affected retinae has an entirely normal spectrum, and presumed from this that the structure is normal. This conclusion makes the necessary assumption that changes in opsin structure would alter the complete optical characteristics of rhodopsin found in the affected retina. This assumption may or may not be correct, since such changes in opsin structure would be likely to concern the make-up of peptide chains in the molecule only. The same authors aiso found that the dystrophic animals had considerably more rhodopsin than corresponding normal controls up to about 30 days of age, after which the rhodopsin concentration in the affected eyes fell rapidly. These authors concluded that the loss of rhodopsin after 30 days of age reflected an inability to resynthesize rhodopsin after bleaching. At first sight the present findings of an early reduction in protein synthesis and Dowling and Sidman’s demonstration of over-production of rhodopsin in dystrophic rats seem irreconcilable, but it should be remembered that here we are measuring the rate of incorporation of amino acid into protein, a measure of net protein synthesis, not a measure of the amount of total retinal protein. It can be seen from the results of the in ri~o experiments that both a depression in rate of protein synthesis and breakdown (i.e. turnover rate) occurs in the dystrophic retina; if a rapid turnover is normal (which it appears to be) then a decrease in this rate is likely to be reflected in an increase in amount of total protein present at any particular instant. It is suggested that the anomaly in protein synthesis in the dystrophic retina results in a reduction in opsin turnover, The increase in total rhodopsin content, as described by Dowiing and Sidman, is possibly a manifestation of a feedback mechanism in an attempt by the affected tissue to conserve rhodopsin. The dramatic drop in rhodopsin content at about 30 days could be the point of breakdown of such a feedback mechanism. Such an imbalance in the visual cycle in the dystrophic retina is likely to be reflected in changes in dynamically linked mechanisms. Some recent work by FWTTERMAN(1963) may be pertinent to this problem. Futterman demonstrated that mammalian visual cell outer segment preparations can metabolize glucose by the “pentose shunt” pathway as well as by glycolysis, and that the “shunt” is implicated in the visual cycle, inasmuch as the reduced nucleotide co-enzyme NADPHs produced during its operation is the principal agent for the reduction of retinene to vitamin A alcohol following light stimulation. It seems plausibIe, therefore, that alteration in the balance of the visual cycle may be reflected by changes in the NADPHsiNADP ratio with concomitant changes in the operation of the shunt mechanism. Whether such changes occur early or late in the course of the development of the dystrophy remains to be seen. SUMMARY By using Cl” labelled glycine. the rate of protein synthesis and the transport of amino acid have been studied both in vitro and in viva in the retina of normal rats and of rats affected with retinal dystrophy, an inherited condition resembling human retinitis pigmentosa. In v&o studies were carried out on retinae from rats over the age range of 6 to 24 days; in vivo studies were confined to rats aged 8 days. The following results were obtained.
The Metabolism of the Dystrophic Retina-II
219
1. In tpitro dystrophic retinae showed a significantly lower rate of incorporation of glycine into total protein than normal retinae at 6 and 8 days of age. At 10 to 24 days the differences, though present, were less marked, until by 24 days incorporation rates were equal. 3 No differences in amino acid uptake from the incubation medium, nor in transport of -. amino acid into the retinal tissue, between normal and affected retinae. were detected in the early stages, but at 24 days the intracellular concentration of glycine was markedly lowered in dystrophic retinae. 3. These in vitro results were confirmed by measuring the incorporation of Cl1 glycine into retinal protein at various intervals following the subcutaneous injection of the labelled amino acid into live intact 8-day-old litter-mate rats. C.ompared with unaffected rats the dystrophic animals showed a marked diminution in incorporation rate (protein synthesis) and, in addition, a slower rate of protein breakdown. Since amino acid incorporation rates into total protein in the livers of normal and affected rats were equal, it may be taken that the differences observed in the retina were not simply due to differences in strain of animal. As in the in vitro experiments, amino acid transport into the retina was equal in normal and dystrophic animals. 4. In an earlier study we have shown that certain changes in the carbohydrate metabolism of the retina are observed only subsequent to the histological degenerative changes in the rods. The anomalous protein synthesis recorded here occurs some 6-8 days before histological changes can be seen in the dystrophic retina-in fact before the retina has become fully differentiated into its various layers. Ackno,u/edge,llen?s-We
are greatly indebted for loyal technical assistance to Mrs. DIANA B~LT~N and secretarial help.
Mr. IVAN P. GREEN and to Miss E. M. GOWER for painstaking
REFERENCES BASSI, M. and BERNELLI-ZAZZERA, A. (1960). Effects of potassium ions in brain respiration and amino acid incorporation into brain proteins in vitro. Experientia 16, 430. BONTING, S. L., CARRAVAGGIO, L. L. and GOURAS, P. (1961). The rhodopsin cycle in the developing vertebrate retina-l. Relation of rhodopsin content, electroretinogram and rod structure in the rat. Exp. E>v Res. 1, 14-24. BOURNE, M. C.. CAMPBELL, D. A. and TANSLEY, K. (1938). Hereditary dcgcneration of the rat retina. Brit. J. Ophtltal.
22, 613-623.
CAMPBELL, P. N. (1958). Protein synthesis with special reference to growth processes both normal and abnormal. Advances in Cancer Research, V, pp. 98-155. Academic Press, New York. CHAIN, E. B., LARSSON, S. and POCCHIARI, F. (1960). The fate of glucose in different parts of the rabbit brain. Proc. ray. Sot. B152, 283-289. DAWSON, R. M. C., ELLIOT, D. C., ELLIOT, W. H. and JONES,K. M. (1959). Data,for Biorhrrnica/ Research. Clarendon Press, Oxford. DE ROBERTIS, E. (1960). Some observations on the ultrastructure and morphogcnesis of photoreceptors. J. gen. P/rysio/. 43, l-l 3. DOWLING, J. E. and SIDMAN, R. L. (1962). Inherited retinal dystrophy in the rat. J. cc//. Biol. 14, 73-IOV. FRANK, M., CHAIN, E. B.. POCCHIARI, F. and Rossr, C. (1959). A modified apparatus for the quantitative evaluation of radio-paper-chromatograms. Selected Scientific Papers. 131.Slrp. Smiro 2, 75-87. FUTTERMAN.S. (1963). Metabolism of the retina. 111.The rble of reduced triphosphopyridinc nuclcotidc in the visual cycle. J. biol. Chem. 238, 1145-l 150. HARRIS, H. (1963). Garrod’s Inborn Errors ofMetabo/isw, pp. 120-197. Oxford University Press, London. READING. H. W. and SORSBY, A. (1962). The metabolism of the dystrophic retina. I. Comparative studies on the glucose metabolism of the developing rat retina, normal and dystrophic. Vision Rcs. 2. 315-325. RICHTER, D. ( 1955). In Bioclwr~istry of’ fhe dewlophg nenms .swetv, pp. 241-243. Ed. WAELSCH, H. Academic Press. New York. RIGGS, T. R. and WALKER, L. M. (1963). Some relations between active transport of free amino acids into cells and their incorporation into protein. J. biol. C/rem. 238, 2663-2668.
220
H. M’.
READING
ANII
ARWLII
SORSB~
RO~DYN, D. B., SUTTIE, J. W. and WORK, T. S. (1962). Protein synthesis in mitochondria. Blorlrcr~~. J. 83, 2940. SIEKEVITZ, P. (1952). Uptake of radioactive alanine i/t Gtro into the proteins of rat liver fractions. d. hiol. Chew. 195, 549-565. STEINBERG. D. and VAUGHAN, M. (1956). Observations in intracellular protein cat‘~bolism studied ;/I I,;/IYI.
Arch.
Biochem.
Biophys.
65, 93-105.
VAN SLYKE, D. D., MACFADYEN, D. A. and HAMILTON. P. (1941). by titration of the carbon of the carbon dioxide formed in the
The determination
of fret amino acids
reaction with ninhydrin.
J. hiol. C/WW.
141, 671.
R&urn&-La synthese de protkine dans la retine du rat, determini par la vitesse d’incorporation de glycine marquee B Cl” est notamment plus reduit dans le cas de la rktine dystrophique en tours de dCveloppement que dans la rttine normale dCveloppante. Cet anomalie devient apparente 6-8 jours aprts la naissance; c’est g dire 6-8 jours avant I’&idence histologique de d&&ration. Ces decouvertes ne sont nullement d’accord, du point de vue du temps, avec les anomalieb connues de la catabolisme de glucose, qui ne se produisent qu’une fois les changements histologiques sont bien ttablies.
Zusammenfassung-Die
Proteinsynthese in der Netzhaut von Ratten wie sie durch den Einbau von mit Cl4 markiertem Glycin gemessen wird, ist in der sich entwickelnden dystrophischen Netzhaut deutlich geringer als in der sich entwickelnden normalen Retina. Dieses anormale Verhalten wird 6-8 Tage nach der Geburt beobachtet, also etwa 6-8 Tage bevor sich der histologiche Befund der Degenerierung zeigt. Die Ergebnisse stehen beziiglich der zeitlichen Reihenfolge im Gegensatz zu den bekannten Anomalien im Glukose Katabolismus, die erst auftreten, wenn die histologischen Verinderungen schon gut ausgebildet
sind.
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