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Adenylate cyclase-phosphodiesterase system in rat olfactory bulb and cerebral cortex during the postnatal period. Effects of growth restriction
598
Brain Research, 193 (1980)598 603 !(~ Elsevier/North-Holland Biomedical Press
Adenylate cyclase-phosphodiesterase system in rat olfactory bulb and cerebral cortex during the postnatal period. Effects of growth restriction
X. COUSIN and J. L. DAVRAINVILLE* Laboratoire de Physiologie Gdndrale 1I, Universttd de Nancy-L Nancy (France)
(Accepted February 28th, 1980) Key words: adenylate cyclase -- phosphodiesterase -- olfactory bulb -- cerebral cortex - - growth - -
postnatal development
During the early postnatal life the rat brain undergoes an accelerated evolution of interrelated morphological, biochemical and electrophysiological parameters. During this evolution, the brain is most sensitive to the effects of external agents: significant alterations of cellular metabolism and electrophysiological properties in response to neonatal undernutrition have been reported 9. Regional variability of developmental pattern and susceptibility to nutritional deprivation, both in timing and amplitude, is demonstrated in the different parts of the brain s . Cell proliferation and metabolism is modulated by genetic, hormonal and environmental factors; some of these factors may act through cyclic nucleotides. Moreover the role of cyclic nucleotides in neuronal function has been demonstrated is and changes of cyclic nucleotide levels would provide valuable information about changes of cell function. The intracellular levels of cyclic AMP are largely controlled by a balance between the activities of two enzyme systems: adenylate cyclase and phosphodiesterase. The purpose of our work was to provide more information on the effects of undernutrition on maturation of the cyclic AMP system in the rat during the postnatal period. Such determinations have been performed in various brain areas 9, 13,17 but not in olfactory bulb; therefore our experiments were realized on olfactory bulb homogenate and a comparison with cerebral cortex preparation was made. Experiments were conducted on Wistar rats from birth to 35 days of age. In the control group, litter size was adjusted to 6 on the day of parturition and dams fed ad libitum an optimum standard laboratory diet containing 18.5 ~ protein throughout the experimental period. The growth-restricted group consisted of the progeny of dams having 12-15 animals per litter and feeding ad libitum, from parturition and throughout the following 3 weeks, a low-protein (8,% casein) isocaloric purified diet. The milk produced by these malnourished dams has been demonstrated not to differ significantly in quality from that of control rats, but is considerably reduced in volume TM. At 21 days undernourished litters were provided with the control diet (nutritional rehabilitation). * To whom correspondence should be addressed.
599 Animals were killed by decapitation. Olfactory bulbs and cerebral cortex were excised rapidly in a refrigerated chamber, pooled and weighed (at least 200 mg wet tissue). Homogenates were prepared in a Potter-Elvehjem apparatus. Tissue DNA was extracted as recommended by Zamenhof et al. 19 and measured colorimetrically by the diphenylamine reaction 1. Protein was estimated using Folin phenol reagent6. Adenylate cyclase activity was assayed according to a modification of the method of MacDonald 7 as presented in detail in an earlier report 3. Phosphodiesterase activity was carried out at 37 °C in a mixture containing the following final concentrations: 60 mM glycyl-glycinbuffer pH 7.5, 3.6 mM MgSO4 and cyclic AMP. The concentrations of homogenate and incubation times were such that no more than 25 % of the substrate was hydrolyzed during the assay period, cAMP was added at an initial concentration of 5 #M and incubation was carried out for 4 min using 150-200 #g/ml tissue protein; this sampled predominantly the low Km form of the enzyme. For the high Km form, substrate was added at initial concentration of 500/~M, tissue protein concentration was 350-400 /~g/ml and incubation was performed for 6 min. The reaction was achieved by a 3.5 min immersion in a boiling water bath and unhydrolyzed substrate was evaluated as above 7. Two sets of control were run, one containing no enzyme, the other no substrate. Under the different conditions, enzyme activity was verified to be linear with respect to time and homogenate concentration over the range used. All values reported were at least the means of two independent determinations. Statistical analysis was performed using the Student's t-test. With our experimental design undernutrition drastically retarded the growth rate: a 76 % reduction in body weight gain and a 28 % reduction in nervous structure weight gain were noted at 21 days. Growth restriction gradually developed throughout the experimental period, the maximum occurring during the third postnatal week (around 35 % in nervous structures). During the two week rehabilitation period the physical characteristics of the experimental rats improved noticeably but values remained significantly less than those in the control group. DNA content (index of cell number) of the olfactory bulb increased sharply, in the controls, from birth to 21 days and then much more slowly; the relative increase in DNA content of the cerebral cortex was less pronounced but DNA accumulation continued until at least day 35. Inversely, mean cell size, as estimated by protein/DNA ratio, was increased more in cerebral cortex than in olfactory bulb. Growth restriction curtailed the normal increase in DNA content, the deficit being higher in cerebral cortex (38.1%) than in olfactory bulb (23.4 %). During the 2 week recovery period, the DNA content of the olfactory bulb increased slightly compared to the control, whereas the deficit in cerebral cortex was increased (Fig. 1). Protein/DNA ratio was unaffected. The specific activity of adenylate cyclase was measured under two different conditions: basal and stimulated; maximum activity was obtained with 6 mM NaF and 0.1% Triton X-100 included in the basal incubation mixture. The stimulation observed in response to the addition of F- or Triton X-100 alone were presented in an earlier report a. Specific activity of basal adenylate cyclase was low at birth in olfactory bulb homogenate; it was enhanced 11.4-fold during the experimental period, increasing during the first and the third postnatal week; it slightly decreased thereafter.
600
100
80L CEREBRAL CORTEX
>.
6O
E E
OLFACTORY BULB 40
~a
20 Z
L
........
~L_______ 7
I
I
14
21
AGE OF RATS
I
35
(days)
Fig. 1. Evolution of DNA content during development of rat olfactory bulb (0) and cerebral cortex (A), from birth to 35 days of postnatal age. Each point is the mean of 10-15 determinations; closed symbols represent the evolution in control animals, open symbols evolution in growth-restricted rats. Results have been calculated as a percentage of the maximum values observed during the experimental period, viz. 147.3 ± 1.3 pg/2 olfactory bulbs and 739.5 :~ 3.8/~g/structure in cerebral cortex (mean -~: S.E.).
Experiments with cerebral cortex homogenate revealed a basal adenylate cyclase of specific activity 3.6 times higher than that of olfactory bulb homogenate in the neonate; it increased sharply during the first week and then declined; activity became similar in both structures from the twenty-first day onwards. Activity was reduced in undernourished rats; the retardation in enzyme evolution was greatest during the first postnatal week in the cerebral cortex (43.7 ~ inhibition) and during the third week in the olfactory bulb (26.6 ~ inhibition). This retardation was further increased during the rehabilitation period, reaching about 50 ~ in both structures. Addition of 0.1 ~ Triton X-100 + 6 mM N a F to the incubation medium enhanced the basal activity in both structures throughout the experimental period. This stimulated activity may be assumed to represent the total number of catalytic units. Activity reached its peak level at day 21 and was significantly higher (39.6 ~/o)in cerebral cortex. Stabilized values were then maintained in olfactory bulb, whereas a
I 7 14 21 35 1 7 14 21 35
Olfactory bulb
23 i 10 (7) 165 i 22 (9) 139 4- 13 (9) 263 4- 8 (13) 231 ± 17 (9) 82 4- 14 (14) 407 4- 13 (10) 264 4- 16 (14) 3054-26(17) 232 4- 20 (6) 44i 4-
23 (7) 21 (6) 14 (7)** 11 (6)**
229 4- 21 (7)** 247 4- 18 (13) 2 5 7 4 - 9 (7) 118 4- 17 (5)**
139 137 193 112
230 4- 18 (13) 422 4- 15 (10) 880 -4- 15 (10) 958 4- 26 (10) 930 i 21 (12) 221 4- 23 (18) 909 4- 29 (8) 1145 4- 63 (12) 13374-24(11) 1037 d: 45 (12) 460 559 693 610
4444-
20 30 21 23
(14) (14)** (13)** (19)**
Growth-restricted
645 4- 29 (12)** 883 4- 41 (12)** 13334-34(19) 930 4- 29 (13)
Control
Control
Growth-restricted
Stimulated activity
Basal activity
* 0.01 < P < 0.05, Student's t-test. ** P < 0.01, Student's t-test.
Cerebral cortex
Age (days)
Structure
Adenylate cyclase activity (pmol/mg protein/min)
N.D. 24.64 4- 1.61 (4) 26.13 4- 1.43 (5) 31.40 4- 2.26 (6) 31.96 4- 1.56 (6) N.D. 36.76 ± 5.12 (8) 44.69 4- 3.19 (6) 70.934-4.60(6) 72.95 4- 1.65 (6)
Control
High-Kin activity
4- 2.77 (5) -4- 1.86 (7) ± 1.85 (6) 4- 2.08 (6) 22.75 4- 2.38 (9)* 37.19 4- 1.73 (8) 55.704-3.57(6)* 78.35 4- 2.03 (5)
22.70 25.62 30.11 37.40
Growth-restricted
Cyclic AMP phosphodiesterase activity (nmol/mg protein~rain)
The activities of adenylate cyclase and high Km phosphodiesterase were assayed as described in text by a modification of the method of MacDonaldL Data represent means 4- S.E.M. for the number of determinations indicated in parentheses. The differences between control and growth-restricted group are determined by the Student's t-test. N.D., activity not detectable by the method used.
Postnatal development of adenylate cyclase and cyclic AMP-phosphodiesterase activity in rat brain homogenate ; influence of growth-restriction
TABLE I
o~
602 very significant drop was observed in cerebral cortex. Stimulated activity was lowered in both structures in the growth-restricted group; the retardation was highly significant in the cerebral cortex as soon as day 7 (29.0 °Jo) and then declined, the difference being made up at day 21. In olfactory bulb homogenate, the retardation appeared during the second postnatal week, reached 36.5'.!J,i and was maintained in spite of nutritional rehabilitation. The two major forms of cyclic AMP phosphodiesterase activity, one with a high affinity, the other with low affinity, followed different patterns of development. Low Km activity increased slowly from 0.5 to 3.0 nmol cAMP hydrolyzed/mg protein/rain between [ and 35 days of age; values were similar in both structures and evolved similarly in control and growth-restricted rats. The activity of the high Km form (Table I) was not detected with our experimental method at day 1; it increased during the postnatal period. In the olfactory bulb this increase was moderate, it proceeded chiefly over the first postnatal week and was unaffected by underfeeding. Significantly higher values were obtained in cerebral cortex homogenate; activity significantly increased during the first and the third postnatal week. It was then significantly lower in growthrestricted rats (38.1 ',!,'iand 21.5 {'~irespectively). A striking improvement was observed during the recovery period. In summary, the comparison between olfactory bulb and cerebral cortex maturation during the postnatal period revealed striking differences: (1) DNA content in olfactory bulb increased sharply between 1 and 21 days of age, whereas DNA accumulation in the cerebral cortex occurred progressively up to day 35; (2) adenylate cyclase and phosphodiesterase activity reached a maximum at day 21 ; the time course and magnitude of this evolution differed among the structures explored. Enzyme maturation seemed somewhat more precocious (basal adenylate cyclase) and activity levels were frequently higher in cerebral cortex; (3) undernutrition curtailed growth, DNA accumulation and enzyme maturation; retardation in enzyme maturation appeared earlier in cerebral cortex and, according to the enzyme, attained higher levels in olfactory bulb (adenylate cyclase) or in cerebral cortex (phosphodiesterase); and (4) nutritional rehabilitation sometimes partially reversed these effects (phosphodiesterase). The postnatal increase in enzyme activity may be attributed either to a disproportionate development in cell type or cell structure (e.g. synaptic structure) rich in activitya7 or to de novo synthesis of enzyme molecules. Cytosol contains protein components (calcium-dependent modulators) regulating phosphodiesterase and cyclase activity12; ontogenetic evolution might be due to increased amounts of these agents. Furthermore, adenylate cyclase being tightly associated with membranes, its evolution may depend on age-related alterations of membrane structure 4 and composition la. Similarly, perturbations of enzyme activity in the growth-restricted group may be related to modifications at enzyme molecule (decreased rate of enzyme synthesis or reduced number of catalytic units 2) or to changes in its environment (membrane structure and composition 14, or levels of cytosol modulatorsla). A significant increase (80-100 o/ /o) in extracellular-free calcium levels was reported in undernourished ratsS: this excess of calcium activity might modify the cytosolic control of enzyme activity 16 or induce alterations in the physico-chemical properties of membranes 15.
603 l Burton, K., A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-322. 2 Cooper, B., Weinblatt, F. and Gregerman, R. I., Enhanced activity of hormone-sensitive adenylate cyclase during dietary restriction in the rat, J. clin. Invest., 59 (1977) 467-474. 3 Cousin, X. and Davrainville, J. L., Adenylate cyclase in the developing rat cerebral cortex and olfactory bulb, Experientia (Basel), 36 (1980) 90-92. 4 De Laat, S. W., Van der Saag, P. T., Ad Nelemans, S. and Shinitzky, M., Microviscosity changes during differentiation of neuroblastoma cells, Biochim. biophys. Acta (Amst.), 509 (1978) 188-193. 5 Fish, [. and Winick, M., Effect of malnutrition on regional growth of the developing rat brain, Exp. Neurol., 25 (1969) 534-540. 6 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 7 MacDonald, I. A., Differentiation of fluoride-stimulated components of beef brain cortex adenylate cyclase by calcium ions, ethyleneglycol-bis-(fl-aminoethyl ether) N, N'otetraacetic acid and Triton X-100, Biochim. biophys. Acta (Amst.), 397 (1975) 244-253. 8 Math, F. and Davrainville, J. L., Postnatal variations of extracellular free calcium levels in the rat. Influence of undernutrition, Experientia (Basel), 35 (1979) 1355-1356. 9 Morgane, P. J., Miller, M., Kemper, T., Stern, W., Forbes, W., Hall, R., Bronzino, J., Kissane, J., Hawrylewicz, E. and Resnick, O., The effects of protein malnutrition on the developing central nervous system in the rat, Neurosci. Biobehav. Rev., 2 (1978) 137-230. 10 Mueller, A. J. and Cox, W. M., The effects of changes in diet on the volume and composition of rat milk, J. Nutr., 31 (1946) 249-259. 11 Pastuszko, A., Gromek, A., Dabrowiecki, Z. and Olszewska, K., Changes of lipid-protein structure of mitochondrial membranes, from rat brain during development, in normal and pathological conditions, Bull. Acad. poL Sci. Biol., 25 (1977) 487-494. 12 Pichard, A. L. and Cheung, W. Y., Cyclic 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics, J. biol. Chem., 251 (1976) 57265737. 13 Strada, S. J., Uzunov, P. and Weiss, B., Ontogenetic development ofa phosphodiesterase activator and the multiple forms of cyclic AMP phosphodiesterase of rat brain, J. Neuroehem., 23 (1974) 1097-1103. 14 Sun, J. V., Tepperman, H. M. and Tepperman, J., Lipid composition of liver plasma membranes isolated from rats fed a high glucose or a high fat diet, J. Nutr., 109 (1979) 193-201. 15 Tr~iuble, H. and Eibl, H., Electrostatic effects on lipid phase transitions: membrane structure and ionic environment, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 214-219. 16 Wang, J. H. and Desai, R., Modulator binding protein. Bovine brain protein exhibiting the Ca 2÷-, dependent association with the protein modulator of cyclic nucleotide phosphodiesterase J. biol. Chem., 252 (1977) 4175-4184. 17 Weiss, B., Ontogenetic development of adenyl cyclase and phosphodiesterase in rat brain, J. Neurochem., 18 (1971) 469-477. 18 Wiegant, V. M., Cyclic nucleotides in nervous tissue, Brain Res. Bull., 3 (1978) 611-622. 19 Zamenhof, S., Grauel, L., Van Marthens, E. and Stillinger, R. A., Quantitative determination of DNA in preserved brains and brain sections, J. Neurochem., 19 (1972) 61-68.
Brain Research, 193 (1980)598 603 !(~ Elsevier/North-Holland Biomedical Press
Adenylate cyclase-phosphodiesterase system in rat olfactory bulb and cerebral cortex during the postnatal period. Effects of growth restriction
X. COUSIN and J. L. DAVRAINVILLE* Laboratoire de Physiologie Gdndrale 1I, Universttd de Nancy-L Nancy (France)
(Accepted February 28th, 1980) Key words: adenylate cyclase -- phosphodiesterase -- olfactory bulb -- cerebral cortex - - growth - -
postnatal development
During the early postnatal life the rat brain undergoes an accelerated evolution of interrelated morphological, biochemical and electrophysiological parameters. During this evolution, the brain is most sensitive to the effects of external agents: significant alterations of cellular metabolism and electrophysiological properties in response to neonatal undernutrition have been reported 9. Regional variability of developmental pattern and susceptibility to nutritional deprivation, both in timing and amplitude, is demonstrated in the different parts of the brain s . Cell proliferation and metabolism is modulated by genetic, hormonal and environmental factors; some of these factors may act through cyclic nucleotides. Moreover the role of cyclic nucleotides in neuronal function has been demonstrated is and changes of cyclic nucleotide levels would provide valuable information about changes of cell function. The intracellular levels of cyclic AMP are largely controlled by a balance between the activities of two enzyme systems: adenylate cyclase and phosphodiesterase. The purpose of our work was to provide more information on the effects of undernutrition on maturation of the cyclic AMP system in the rat during the postnatal period. Such determinations have been performed in various brain areas 9, 13,17 but not in olfactory bulb; therefore our experiments were realized on olfactory bulb homogenate and a comparison with cerebral cortex preparation was made. Experiments were conducted on Wistar rats from birth to 35 days of age. In the control group, litter size was adjusted to 6 on the day of parturition and dams fed ad libitum an optimum standard laboratory diet containing 18.5 ~ protein throughout the experimental period. The growth-restricted group consisted of the progeny of dams having 12-15 animals per litter and feeding ad libitum, from parturition and throughout the following 3 weeks, a low-protein (8,% casein) isocaloric purified diet. The milk produced by these malnourished dams has been demonstrated not to differ significantly in quality from that of control rats, but is considerably reduced in volume TM. At 21 days undernourished litters were provided with the control diet (nutritional rehabilitation). * To whom correspondence should be addressed.
599 Animals were killed by decapitation. Olfactory bulbs and cerebral cortex were excised rapidly in a refrigerated chamber, pooled and weighed (at least 200 mg wet tissue). Homogenates were prepared in a Potter-Elvehjem apparatus. Tissue DNA was extracted as recommended by Zamenhof et al. 19 and measured colorimetrically by the diphenylamine reaction 1. Protein was estimated using Folin phenol reagent6. Adenylate cyclase activity was assayed according to a modification of the method of MacDonald 7 as presented in detail in an earlier report 3. Phosphodiesterase activity was carried out at 37 °C in a mixture containing the following final concentrations: 60 mM glycyl-glycinbuffer pH 7.5, 3.6 mM MgSO4 and cyclic AMP. The concentrations of homogenate and incubation times were such that no more than 25 % of the substrate was hydrolyzed during the assay period, cAMP was added at an initial concentration of 5 #M and incubation was carried out for 4 min using 150-200 #g/ml tissue protein; this sampled predominantly the low Km form of the enzyme. For the high Km form, substrate was added at initial concentration of 500/~M, tissue protein concentration was 350-400 /~g/ml and incubation was performed for 6 min. The reaction was achieved by a 3.5 min immersion in a boiling water bath and unhydrolyzed substrate was evaluated as above 7. Two sets of control were run, one containing no enzyme, the other no substrate. Under the different conditions, enzyme activity was verified to be linear with respect to time and homogenate concentration over the range used. All values reported were at least the means of two independent determinations. Statistical analysis was performed using the Student's t-test. With our experimental design undernutrition drastically retarded the growth rate: a 76 % reduction in body weight gain and a 28 % reduction in nervous structure weight gain were noted at 21 days. Growth restriction gradually developed throughout the experimental period, the maximum occurring during the third postnatal week (around 35 % in nervous structures). During the two week rehabilitation period the physical characteristics of the experimental rats improved noticeably but values remained significantly less than those in the control group. DNA content (index of cell number) of the olfactory bulb increased sharply, in the controls, from birth to 21 days and then much more slowly; the relative increase in DNA content of the cerebral cortex was less pronounced but DNA accumulation continued until at least day 35. Inversely, mean cell size, as estimated by protein/DNA ratio, was increased more in cerebral cortex than in olfactory bulb. Growth restriction curtailed the normal increase in DNA content, the deficit being higher in cerebral cortex (38.1%) than in olfactory bulb (23.4 %). During the 2 week recovery period, the DNA content of the olfactory bulb increased slightly compared to the control, whereas the deficit in cerebral cortex was increased (Fig. 1). Protein/DNA ratio was unaffected. The specific activity of adenylate cyclase was measured under two different conditions: basal and stimulated; maximum activity was obtained with 6 mM NaF and 0.1% Triton X-100 included in the basal incubation mixture. The stimulation observed in response to the addition of F- or Triton X-100 alone were presented in an earlier report a. Specific activity of basal adenylate cyclase was low at birth in olfactory bulb homogenate; it was enhanced 11.4-fold during the experimental period, increasing during the first and the third postnatal week; it slightly decreased thereafter.
600
100
80L CEREBRAL CORTEX
>.
6O
E E
OLFACTORY BULB 40
~a
20 Z
L
........
~L_______ 7
I
I
14
21
AGE OF RATS
I
35
(days)
Fig. 1. Evolution of DNA content during development of rat olfactory bulb (0) and cerebral cortex (A), from birth to 35 days of postnatal age. Each point is the mean of 10-15 determinations; closed symbols represent the evolution in control animals, open symbols evolution in growth-restricted rats. Results have been calculated as a percentage of the maximum values observed during the experimental period, viz. 147.3 ± 1.3 pg/2 olfactory bulbs and 739.5 :~ 3.8/~g/structure in cerebral cortex (mean -~: S.E.).
Experiments with cerebral cortex homogenate revealed a basal adenylate cyclase of specific activity 3.6 times higher than that of olfactory bulb homogenate in the neonate; it increased sharply during the first week and then declined; activity became similar in both structures from the twenty-first day onwards. Activity was reduced in undernourished rats; the retardation in enzyme evolution was greatest during the first postnatal week in the cerebral cortex (43.7 ~ inhibition) and during the third week in the olfactory bulb (26.6 ~ inhibition). This retardation was further increased during the rehabilitation period, reaching about 50 ~ in both structures. Addition of 0.1 ~ Triton X-100 + 6 mM N a F to the incubation medium enhanced the basal activity in both structures throughout the experimental period. This stimulated activity may be assumed to represent the total number of catalytic units. Activity reached its peak level at day 21 and was significantly higher (39.6 ~/o)in cerebral cortex. Stabilized values were then maintained in olfactory bulb, whereas a
I 7 14 21 35 1 7 14 21 35
Olfactory bulb
23 i 10 (7) 165 i 22 (9) 139 4- 13 (9) 263 4- 8 (13) 231 ± 17 (9) 82 4- 14 (14) 407 4- 13 (10) 264 4- 16 (14) 3054-26(17) 232 4- 20 (6) 44i 4-
23 (7) 21 (6) 14 (7)** 11 (6)**
229 4- 21 (7)** 247 4- 18 (13) 2 5 7 4 - 9 (7) 118 4- 17 (5)**
139 137 193 112
230 4- 18 (13) 422 4- 15 (10) 880 -4- 15 (10) 958 4- 26 (10) 930 i 21 (12) 221 4- 23 (18) 909 4- 29 (8) 1145 4- 63 (12) 13374-24(11) 1037 d: 45 (12) 460 559 693 610
4444-
20 30 21 23
(14) (14)** (13)** (19)**
Growth-restricted
645 4- 29 (12)** 883 4- 41 (12)** 13334-34(19) 930 4- 29 (13)
Control
Control
Growth-restricted
Stimulated activity
Basal activity
* 0.01 < P < 0.05, Student's t-test. ** P < 0.01, Student's t-test.
Cerebral cortex
Age (days)
Structure
Adenylate cyclase activity (pmol/mg protein/min)
N.D. 24.64 4- 1.61 (4) 26.13 4- 1.43 (5) 31.40 4- 2.26 (6) 31.96 4- 1.56 (6) N.D. 36.76 ± 5.12 (8) 44.69 4- 3.19 (6) 70.934-4.60(6) 72.95 4- 1.65 (6)
Control
High-Kin activity
4- 2.77 (5) -4- 1.86 (7) ± 1.85 (6) 4- 2.08 (6) 22.75 4- 2.38 (9)* 37.19 4- 1.73 (8) 55.704-3.57(6)* 78.35 4- 2.03 (5)
22.70 25.62 30.11 37.40
Growth-restricted
Cyclic AMP phosphodiesterase activity (nmol/mg protein~rain)
The activities of adenylate cyclase and high Km phosphodiesterase were assayed as described in text by a modification of the method of MacDonaldL Data represent means 4- S.E.M. for the number of determinations indicated in parentheses. The differences between control and growth-restricted group are determined by the Student's t-test. N.D., activity not detectable by the method used.
Postnatal development of adenylate cyclase and cyclic AMP-phosphodiesterase activity in rat brain homogenate ; influence of growth-restriction
TABLE I
o~
602 very significant drop was observed in cerebral cortex. Stimulated activity was lowered in both structures in the growth-restricted group; the retardation was highly significant in the cerebral cortex as soon as day 7 (29.0 °Jo) and then declined, the difference being made up at day 21. In olfactory bulb homogenate, the retardation appeared during the second postnatal week, reached 36.5'.!J,i and was maintained in spite of nutritional rehabilitation. The two major forms of cyclic AMP phosphodiesterase activity, one with a high affinity, the other with low affinity, followed different patterns of development. Low Km activity increased slowly from 0.5 to 3.0 nmol cAMP hydrolyzed/mg protein/rain between [ and 35 days of age; values were similar in both structures and evolved similarly in control and growth-restricted rats. The activity of the high Km form (Table I) was not detected with our experimental method at day 1; it increased during the postnatal period. In the olfactory bulb this increase was moderate, it proceeded chiefly over the first postnatal week and was unaffected by underfeeding. Significantly higher values were obtained in cerebral cortex homogenate; activity significantly increased during the first and the third postnatal week. It was then significantly lower in growthrestricted rats (38.1 ',!,'iand 21.5 {'~irespectively). A striking improvement was observed during the recovery period. In summary, the comparison between olfactory bulb and cerebral cortex maturation during the postnatal period revealed striking differences: (1) DNA content in olfactory bulb increased sharply between 1 and 21 days of age, whereas DNA accumulation in the cerebral cortex occurred progressively up to day 35; (2) adenylate cyclase and phosphodiesterase activity reached a maximum at day 21 ; the time course and magnitude of this evolution differed among the structures explored. Enzyme maturation seemed somewhat more precocious (basal adenylate cyclase) and activity levels were frequently higher in cerebral cortex; (3) undernutrition curtailed growth, DNA accumulation and enzyme maturation; retardation in enzyme maturation appeared earlier in cerebral cortex and, according to the enzyme, attained higher levels in olfactory bulb (adenylate cyclase) or in cerebral cortex (phosphodiesterase); and (4) nutritional rehabilitation sometimes partially reversed these effects (phosphodiesterase). The postnatal increase in enzyme activity may be attributed either to a disproportionate development in cell type or cell structure (e.g. synaptic structure) rich in activitya7 or to de novo synthesis of enzyme molecules. Cytosol contains protein components (calcium-dependent modulators) regulating phosphodiesterase and cyclase activity12; ontogenetic evolution might be due to increased amounts of these agents. Furthermore, adenylate cyclase being tightly associated with membranes, its evolution may depend on age-related alterations of membrane structure 4 and composition la. Similarly, perturbations of enzyme activity in the growth-restricted group may be related to modifications at enzyme molecule (decreased rate of enzyme synthesis or reduced number of catalytic units 2) or to changes in its environment (membrane structure and composition 14, or levels of cytosol modulatorsla). A significant increase (80-100 o/ /o) in extracellular-free calcium levels was reported in undernourished ratsS: this excess of calcium activity might modify the cytosolic control of enzyme activity 16 or induce alterations in the physico-chemical properties of membranes 15.
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