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Development of phosphodiesterase activity in the chick retina
DEVELOPMENTAL
BIOLOGY
Development
40, 378-380 (1974)
of Phosphodiesterase
Activity
in the Chick Retina
GERALD J. CHADER, R. T. FLETCHER, AND DAVID A. NEWSOME Laboratory
of Vision Research,
Health,
National Eye Institute, National Institutes of Health, Education, and Welfare, Bethesda, Maryland 20014 Accepted
U.S. Department
of
June 17, 1974
Significant cyclic GMP phosphodiesterase activity is apparent in the early stages of development of the chick neural retina. By day 8, specific activity drops by approximately two-thirds. After day 14, a sharp rise in activity is observed, continuing through the time of hatching. Cyclic AMP phbsphodiesterase activity is considerably lower and does not markedly change over the embryonic period.
The neural retina of the chick embryo is an excellent model for the study of early morphological and biochemical development of a tissue (Coulombre, 1955). Structural and enzymatic patterns can be correlated in an attempt to determine possible temporal relationships between these changes. The enzymes of cyclic nucleotide synthesis and metabolism have recently been shown to be present in the retina. In particular, the activity of phosphodiesterase, the enzyme(s) of cyclic nucleotide hydrolysis, has been shown to be extremely high in retina (Pannbacker et al., 1972; Chader et al., 1974) making it of interest to study its development in relation to other ontogenic changes. In the present study we report on the development of hydrolytic activity for cyclic AMP and cyclic GMP in the chick embryo retina and discuss these changes in relation to other events occurring at this time. MATERIALS
AND
METHODS
Embryos of White Leghorn eggs (Truslow Farms, Chesterton, Maryland) were staged according to the criteria of Hamburger and Hamilton (1951). Cleanly dissected neural retinas were washed in 40 mM Tris buffer, pH 7.6, containing 5 mM MgC12, and gently homogenized in a glassTeflon homogenizer. Phosphodiesterase determinations were performed in dupli-
cate on at least 3 retinas obtained from separate embryos aged 9 days or older. For younger embryos, several retinas were pooled before homogenization to afford enough material for the 3 separate samples. Duplicate assays were then performed on each of the 3 pooled samples. Assay for phosphodiesterase activity was by the method of Thompson and Appleman (1971). Assay was for 10 min at 25” with a substrate concentration of 5 &4 cyclic nucleotide. Micromolar concentrations of substrate were used in the present study, concentrations which approach physiological levels of the cyclic nucleotides in uiuo. The conditions used were essentially those of Beavo et al. (1970) with a slight modification (Chader et al., 1974). The retinal phosphodiesterase preparations were suitably diluted to obtain approximately 15-20% substrate utilization.. Nucleotide hydrolysis was linear with time for at least 15 min under these conditions. Phosphodiesterase activity is expressed as micromoles of cyclic nucleotide hydrolyzed per milligram of protein per minute at 25”. Radiolabeled cyclic AMP (22.1 Ci/mmole) and cyclic GMP (3.5 Ci/mmole) were purchased from New England Nuclear, Boston, Massachusetts; other chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri. Protein was determined by the method of Lowry et al. (1951).
378 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
BRIEF NOTES RESULTS
AND DISCUSSION
Figure 1 shows the developmental pattern of cyclic GMP and cyclic AMP phosphodiesterase activity in the chick retina from stage 18 (about 3 days in ouo) to just before hatching at day 21. Specific activity of cyclic GMP hydrolysis is high to approximately stage 26 after which activity decreases to about day 9. A slow increase in activity is then observed until day 15, when it rises sharply to the time of hatching. Cyclic AMP hydrolytic activity is considerably lower than that for cyclic GMP. Moreover, there is little change in specific activity throughout the period of embryonic development although the same general trend as with cyclic GMP is observed. It appears from recent work that cyclic nucleotide phosphodiesterase is not a single enzyme, but rather a complex class of enzymes, existing in distinct molecular and kinetic forms (Appleman et al., 1973). Forms of phosphodiesterase separated from brain, for example, exhibit several molecular weights, have different substrate specificities and also multiple Michaelis constants (Thompson and Appleman, 7 5 15! P
*Cycle GMP o Cyclic AMP
I
r
‘/
/’
g
L-
,
I8 2
24 29 STAGE
-L1
f‘,
32
I
~1
IO
1
1
I~-,
L-i
/
12 14 I6 18 DAYS OF AGE I mow)
20
FIG. 1. Development of cyclic GMP and cyclic AMP phosphodiesterase activity in the chick embryo retina. Values given are averages from triplicate samples as described in the text from at least two separate experiments. Replicate agreement was within 10%. Phosphodiesterase activity is expressed as nmoles of cyclic nucleotide hydrolyzed per milligram of protein per minute.
379
1971). In particular, Sutherland and his co-workers (Beavo et al. 1970, 1971) and Thompson and Appleman (1971) have used micromolar concentrations of cyclic nucleotides to study the kinetic properties of the enzyme(s). Under these more physiological conditions, marked differences in the ratios of hydrolysis of cyclic AMP and cyclic GMP in various tissues have been observed (Beavo et al., 1970) and also stimulatory and inhibitory effects of cyclic GMP on cyclic AMP hydrolysis is apparent (Beavo et al., 1971). Perhaps of greatest importance, multiple kinetic forms (Km) of the enzyme have been elucidated (Russell et al., 1973). Use of such nonsaturating concentrations of substrate focuses on the importance of Km values as a more significant kinetic parameter than V,,,,, and potentially discerns “real” enzyme activities from idealized ones obtained at millimolar concentrations of substrate. The adult bovine retina has also been found to exhibit multiple kinetic forms of the phosphodiesterase enzyme and altered cyclic AMP hydrolysis in the presence of cyclic GMP (Chader et al., 1974). Invagination of the chick optic vesicle occurs at stage 12-14. By stage 18-19, the neural retina is already multilayered and ganglion cell axons have reached the choroid fissure (Goldberg and Coulombre, 1972). The early high specific activity of the phosphodiesterase enzyme appears at a time of ganglionic development, the appearance of Mueller fibers, and preliminary organization of the retina. Days 3-7 (stages 19-34) is a period of log growth of the retina with an increase in thickness from approximately 7 layers of cells to 16 layers by stage 34 (Coulombre, 1955). This corresponds to an approximately 3-fold decrease in cyclic GMP hydrolytic activity. It has been postulated that increased cyclic GMP levels may be involved in the initiation of events leading to cell division (Hadden et al., 1972). Decreased cyclic GMP
380
DEVELOPMENTALBIOLOGY
phosphodiesterase activity as seen in the present study, could lead to increased cyclic GMP levels in the retina, thus signaling for cell proliferation. Alternatively, an increase in cell number at this time could “dilute” the phosphodiesterase enzyme activity if synthesis of the specific enzyme was less rapid than the increase in general protein content or if there were a preferential proliferation of cell types having lower phosphodiesterase activity. The slow increase in phosphodiesterase activity after day 9 corresponds temporally to the development of the outer plexiform layer and the appearance of the photoreceptor inner segments which increase in size to day 15. At day 15, the outer segments appear; elongation occurs to approximately day 19, when this and other components of the retina are functionally complete (Coulombre, 1955). Since cyclic GMP phosphodiesterase activity also abruptly increases at day 15, it is tempting to postulate a correlation between increased phosphodiesterase activity and rod outer segment development. It has been shown that isolated bovine outer segments have extremely high phosphodiesterase activity that seems to be specific for cyclic GMP (Pannbacker et al., 1972; Chader et al., 1974) and that hydrolytic activity is considerably higher in outer segments than in other subcellular fractions of the retina (Chader et al., 1974). Light-sensitive guanylate cyclase activity has also been localized in rod outer segments (Pannbacker, 1973; Bensinger et al., 1974). It thus may be that the greatly increased activity of phosphodiesterase after day 15 of development of the chick may be compartmentalized in the photoreceptor unit. In preliminary studies, we have found that cyclic nucleotide synthetic capability in the chick retina also increases markedly at this time. This could be a most interesting system, then, for studying both morphological and biochemical aspects of the development of
VOLUME 40, 1974
the retina and the photoreceptor unit, especially with regard to the possible role of cyclic nucleotides in vision. REFERENCES APPLEMAN, M., THOMPSON, W., and RUSSELL, T. (1973). Aduan. Cyclic Nucleotide Res. 3, 65-98. BEAVO, J., HARDMAN, J., and SUTHERLAND, E. (1970). Hydrolysis of cyclic guanosine and adenosine 3’) 5’. monophosphates by rat and bovine tissues. J. Biol. Chem. 245, 5649-5655. BEAVO, J., HARDMAN, J., and SUTHERLAND, E. (1971). Stimulation of adenosine 3’,5’-monophosphate hydrolysis by guanosine 3’,5’-monophosphate. J. Biol. Chem. 246, 3841-3846. BENSINGER, R., FLETCHER, R., and CHADER, G. (1974). Guanylate cyclase: inhibition by light in retinal photoreceptors. Science 183, 86-87. CHADER, G., JOHNSON, M., FLETCHER, R., and BENSINGER, R. (1974). Cyclic nucleotide phosphodiesterase of the bovine retina: activity, subcellular distribution and kinetic parameters. J. Neurochem. 22, 93-99. COULOMBRE, A. J. (1955). Correlations of structural and biochemical changes in the developing retina of the chick. Amer. J. Anat. 96, 153-193. GOLDBERG,S., and COULOMBRE,A. (1972). Topographical development of the ganglion cell fiber layer in the chick retina. A whole mount study. J. Comp. Neurol. 146, 507-517. HADDEN, J., HADDEN, E., HADDOX, M., and GOLDBERG, N. (1972). Guanosine 3’) 5’-cyclic monophosphate: A possible intracellular initiator of mitogenic influences in lymphocytes. Proc. Nat. Acad. Sci. U.S. 69, 3024-3027. HAMBURGER, V., and HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-74. LOWRY, O., ROSEBROUGH,N., FARR, A., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. PANNBACKER, R. (1973). Control of guanylate cyclase in the rod outer segment. Science 182, 1138-1140. PANNBACKER,R., FLEISCHMAN, D., and REED, D. (1972). Cyclic nucleotide phosphodiesterase: high activity in a mammalian photoreceptor. Science 175, 757-758. RUSSELL, T., TERASAKI, W., and APPLEMAN, M. (1973). Separate phosphodiesterases for the hydrolysis of cyclic adenosine 3’,5’-monophosphate and cyclic guanosine 3’,5’-monophosphate in rat liver. J. Biol. Chem. 248, 1334-1340. THOMPSON, W., and APPLEMAN, M. (1971). Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry 10, 311-316.
BIOLOGY
Development
40, 378-380 (1974)
of Phosphodiesterase
Activity
in the Chick Retina
GERALD J. CHADER, R. T. FLETCHER, AND DAVID A. NEWSOME Laboratory
of Vision Research,
Health,
National Eye Institute, National Institutes of Health, Education, and Welfare, Bethesda, Maryland 20014 Accepted
U.S. Department
of
June 17, 1974
Significant cyclic GMP phosphodiesterase activity is apparent in the early stages of development of the chick neural retina. By day 8, specific activity drops by approximately two-thirds. After day 14, a sharp rise in activity is observed, continuing through the time of hatching. Cyclic AMP phbsphodiesterase activity is considerably lower and does not markedly change over the embryonic period.
The neural retina of the chick embryo is an excellent model for the study of early morphological and biochemical development of a tissue (Coulombre, 1955). Structural and enzymatic patterns can be correlated in an attempt to determine possible temporal relationships between these changes. The enzymes of cyclic nucleotide synthesis and metabolism have recently been shown to be present in the retina. In particular, the activity of phosphodiesterase, the enzyme(s) of cyclic nucleotide hydrolysis, has been shown to be extremely high in retina (Pannbacker et al., 1972; Chader et al., 1974) making it of interest to study its development in relation to other ontogenic changes. In the present study we report on the development of hydrolytic activity for cyclic AMP and cyclic GMP in the chick embryo retina and discuss these changes in relation to other events occurring at this time. MATERIALS
AND
METHODS
Embryos of White Leghorn eggs (Truslow Farms, Chesterton, Maryland) were staged according to the criteria of Hamburger and Hamilton (1951). Cleanly dissected neural retinas were washed in 40 mM Tris buffer, pH 7.6, containing 5 mM MgC12, and gently homogenized in a glassTeflon homogenizer. Phosphodiesterase determinations were performed in dupli-
cate on at least 3 retinas obtained from separate embryos aged 9 days or older. For younger embryos, several retinas were pooled before homogenization to afford enough material for the 3 separate samples. Duplicate assays were then performed on each of the 3 pooled samples. Assay for phosphodiesterase activity was by the method of Thompson and Appleman (1971). Assay was for 10 min at 25” with a substrate concentration of 5 &4 cyclic nucleotide. Micromolar concentrations of substrate were used in the present study, concentrations which approach physiological levels of the cyclic nucleotides in uiuo. The conditions used were essentially those of Beavo et al. (1970) with a slight modification (Chader et al., 1974). The retinal phosphodiesterase preparations were suitably diluted to obtain approximately 15-20% substrate utilization.. Nucleotide hydrolysis was linear with time for at least 15 min under these conditions. Phosphodiesterase activity is expressed as micromoles of cyclic nucleotide hydrolyzed per milligram of protein per minute at 25”. Radiolabeled cyclic AMP (22.1 Ci/mmole) and cyclic GMP (3.5 Ci/mmole) were purchased from New England Nuclear, Boston, Massachusetts; other chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri. Protein was determined by the method of Lowry et al. (1951).
378 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
BRIEF NOTES RESULTS
AND DISCUSSION
Figure 1 shows the developmental pattern of cyclic GMP and cyclic AMP phosphodiesterase activity in the chick retina from stage 18 (about 3 days in ouo) to just before hatching at day 21. Specific activity of cyclic GMP hydrolysis is high to approximately stage 26 after which activity decreases to about day 9. A slow increase in activity is then observed until day 15, when it rises sharply to the time of hatching. Cyclic AMP hydrolytic activity is considerably lower than that for cyclic GMP. Moreover, there is little change in specific activity throughout the period of embryonic development although the same general trend as with cyclic GMP is observed. It appears from recent work that cyclic nucleotide phosphodiesterase is not a single enzyme, but rather a complex class of enzymes, existing in distinct molecular and kinetic forms (Appleman et al., 1973). Forms of phosphodiesterase separated from brain, for example, exhibit several molecular weights, have different substrate specificities and also multiple Michaelis constants (Thompson and Appleman, 7 5 15! P
*Cycle GMP o Cyclic AMP
I
r
‘/
/’
g
L-
,
I8 2
24 29 STAGE
-L1
f‘,
32
I
~1
IO
1
1
I~-,
L-i
/
12 14 I6 18 DAYS OF AGE I mow)
20
FIG. 1. Development of cyclic GMP and cyclic AMP phosphodiesterase activity in the chick embryo retina. Values given are averages from triplicate samples as described in the text from at least two separate experiments. Replicate agreement was within 10%. Phosphodiesterase activity is expressed as nmoles of cyclic nucleotide hydrolyzed per milligram of protein per minute.
379
1971). In particular, Sutherland and his co-workers (Beavo et al. 1970, 1971) and Thompson and Appleman (1971) have used micromolar concentrations of cyclic nucleotides to study the kinetic properties of the enzyme(s). Under these more physiological conditions, marked differences in the ratios of hydrolysis of cyclic AMP and cyclic GMP in various tissues have been observed (Beavo et al., 1970) and also stimulatory and inhibitory effects of cyclic GMP on cyclic AMP hydrolysis is apparent (Beavo et al., 1971). Perhaps of greatest importance, multiple kinetic forms (Km) of the enzyme have been elucidated (Russell et al., 1973). Use of such nonsaturating concentrations of substrate focuses on the importance of Km values as a more significant kinetic parameter than V,,,,, and potentially discerns “real” enzyme activities from idealized ones obtained at millimolar concentrations of substrate. The adult bovine retina has also been found to exhibit multiple kinetic forms of the phosphodiesterase enzyme and altered cyclic AMP hydrolysis in the presence of cyclic GMP (Chader et al., 1974). Invagination of the chick optic vesicle occurs at stage 12-14. By stage 18-19, the neural retina is already multilayered and ganglion cell axons have reached the choroid fissure (Goldberg and Coulombre, 1972). The early high specific activity of the phosphodiesterase enzyme appears at a time of ganglionic development, the appearance of Mueller fibers, and preliminary organization of the retina. Days 3-7 (stages 19-34) is a period of log growth of the retina with an increase in thickness from approximately 7 layers of cells to 16 layers by stage 34 (Coulombre, 1955). This corresponds to an approximately 3-fold decrease in cyclic GMP hydrolytic activity. It has been postulated that increased cyclic GMP levels may be involved in the initiation of events leading to cell division (Hadden et al., 1972). Decreased cyclic GMP
380
DEVELOPMENTALBIOLOGY
phosphodiesterase activity as seen in the present study, could lead to increased cyclic GMP levels in the retina, thus signaling for cell proliferation. Alternatively, an increase in cell number at this time could “dilute” the phosphodiesterase enzyme activity if synthesis of the specific enzyme was less rapid than the increase in general protein content or if there were a preferential proliferation of cell types having lower phosphodiesterase activity. The slow increase in phosphodiesterase activity after day 9 corresponds temporally to the development of the outer plexiform layer and the appearance of the photoreceptor inner segments which increase in size to day 15. At day 15, the outer segments appear; elongation occurs to approximately day 19, when this and other components of the retina are functionally complete (Coulombre, 1955). Since cyclic GMP phosphodiesterase activity also abruptly increases at day 15, it is tempting to postulate a correlation between increased phosphodiesterase activity and rod outer segment development. It has been shown that isolated bovine outer segments have extremely high phosphodiesterase activity that seems to be specific for cyclic GMP (Pannbacker et al., 1972; Chader et al., 1974) and that hydrolytic activity is considerably higher in outer segments than in other subcellular fractions of the retina (Chader et al., 1974). Light-sensitive guanylate cyclase activity has also been localized in rod outer segments (Pannbacker, 1973; Bensinger et al., 1974). It thus may be that the greatly increased activity of phosphodiesterase after day 15 of development of the chick may be compartmentalized in the photoreceptor unit. In preliminary studies, we have found that cyclic nucleotide synthetic capability in the chick retina also increases markedly at this time. This could be a most interesting system, then, for studying both morphological and biochemical aspects of the development of
VOLUME 40, 1974
the retina and the photoreceptor unit, especially with regard to the possible role of cyclic nucleotides in vision. REFERENCES APPLEMAN, M., THOMPSON, W., and RUSSELL, T. (1973). Aduan. Cyclic Nucleotide Res. 3, 65-98. BEAVO, J., HARDMAN, J., and SUTHERLAND, E. (1970). Hydrolysis of cyclic guanosine and adenosine 3’) 5’. monophosphates by rat and bovine tissues. J. Biol. Chem. 245, 5649-5655. BEAVO, J., HARDMAN, J., and SUTHERLAND, E. (1971). Stimulation of adenosine 3’,5’-monophosphate hydrolysis by guanosine 3’,5’-monophosphate. J. Biol. Chem. 246, 3841-3846. BENSINGER, R., FLETCHER, R., and CHADER, G. (1974). Guanylate cyclase: inhibition by light in retinal photoreceptors. Science 183, 86-87. CHADER, G., JOHNSON, M., FLETCHER, R., and BENSINGER, R. (1974). Cyclic nucleotide phosphodiesterase of the bovine retina: activity, subcellular distribution and kinetic parameters. J. Neurochem. 22, 93-99. COULOMBRE, A. J. (1955). Correlations of structural and biochemical changes in the developing retina of the chick. Amer. J. Anat. 96, 153-193. GOLDBERG,S., and COULOMBRE,A. (1972). Topographical development of the ganglion cell fiber layer in the chick retina. A whole mount study. J. Comp. Neurol. 146, 507-517. HADDEN, J., HADDEN, E., HADDOX, M., and GOLDBERG, N. (1972). Guanosine 3’) 5’-cyclic monophosphate: A possible intracellular initiator of mitogenic influences in lymphocytes. Proc. Nat. Acad. Sci. U.S. 69, 3024-3027. HAMBURGER, V., and HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-74. LOWRY, O., ROSEBROUGH,N., FARR, A., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. PANNBACKER, R. (1973). Control of guanylate cyclase in the rod outer segment. Science 182, 1138-1140. PANNBACKER,R., FLEISCHMAN, D., and REED, D. (1972). Cyclic nucleotide phosphodiesterase: high activity in a mammalian photoreceptor. Science 175, 757-758. RUSSELL, T., TERASAKI, W., and APPLEMAN, M. (1973). Separate phosphodiesterases for the hydrolysis of cyclic adenosine 3’,5’-monophosphate and cyclic guanosine 3’,5’-monophosphate in rat liver. J. Biol. Chem. 248, 1334-1340. THOMPSON, W., and APPLEMAN, M. (1971). Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry 10, 311-316.