THE EFFECT OF NEONATAL THYROIDECTOMY ON THE DEVELOPMENT OF THE ADENOSINE 3'',5''-MONOPHOSPHATE SYSTEM IN THE RAT BRAIN

Abstract— The effect of neonatal thyroidectomy on the cyclic AMP system in the developing rat brain was examined. Administration of ¹³¹I at birth led to a 16 per cent reduction in brain weight and a 70 per cent reduction in body weight by 40 days of age. The level of cyclic AMP in the brain increased 5-fold between birth and 40 days of age and this increase was partially reduced by early thyroidectomy. A similar increase in the activity of adenyl cyclase and phosphodiesterase was observed during development, but thyroidectomy produced no detectable changes in the activity of either enzyme. The activity of the cyclic AMP-dependent protein kinase was already maximal at birth and also was unaffected by thyroidectomy.
Norepinephrine increased levels of cyclic AMP 4- to 5-fold in brain slices prepared from adult rats, but was without effect on slices prepared from newborn or 3-day-old rats. The response to norepinephrine in thyroidectomized rats did not differ from that in control rats at any of the ages examined. Our findings indicate that neonatal hypothyroidism does not deleteriously affect the development of the cyclic AMP system in the rat brain.     

Developmental increase in the inotropic and cyclic AMP response to isoproterenol in embryonic and newly hatched chicks

The cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels of ventricles isolated from 15- to 20-day-old chick embryos and 0- to 3-day-old hatched chicks were compared to clarify the mechanism underlying the change in sensitivity to isoproterenol during perinatal developmental stages when the functional sympathetic innervation has been completely achieved. Isoproterenol produced a positive inotropic effect on ventricles isolated from both embryonic and hatched chicks, but the ventricles from the hatched chicks were more sensitive. At both developmental stages sotalol was an equipotent antagonist of isoproterenol. 3-Isobutyl-1-methylxanthine (IBMX) produced an increment in the contractile force of the ventricles at both stages, but the ventricles from the hatched chicks responded to lower doses of IBMX. The reactivity to isoproterenol in increasing cyclic AMP level was significantly higher in the hatched ventricles than in the embryonic ventricles. The results suggest that the different sensitivities to isoproterenol between embryonic and newly hatched chick ventricles may be due to some changes in the process for cyclic AMP production.     

Oligodendroglial Signal Transduction Systems Are Developmentally Regulated

Abstract: Studies from several laboratories indicate that oligodendroglia exhibit signal transduction systems that can be activated by classical neurotransmitters. Previous studies from this laboratory indicate that oligodendroglia express neuroligand receptors linked to the regulation of Ca²⁺_i. Experiments presented in this article were designed to determine if developmental processes that influence the ability of oligodendroglia to respond to neuroligands with an increase in Ca²⁺_i proceed either in vitro or in vivo. Findings support the view that developmental processes markedly affected the sensitivity of these cells to both purinergic and cholinergic receptor agonists, whereas their responsiveness to either histamine or bradykinin appeared relatively stable over time. Approximately 90 and 75% of oligodendroglia responded to ATP or carbachol, respectively, after 4 days in vitro, whereas <10% of these cells responded to either of these neuroligands after 8 days in vitro. The decrease in the percentage of oligodendroglia responding to ATP, but not carbachol, could be prevented by including dibutyryl cyclic AMP in the culture medium during the final 4 days in vitro. However, once the loss in responsiveness to ATP had occurred, it could not be reversed by exposure to dibutyryl cyclic AMP. Developmental changes in the ATP sensitivity of oligodendroglia occurred in cells expressing galactocerebroside and myelin basic protein. The neuroligand sensitivity of oligodendroglia isolated from either neonatal, 2-, 3-, or 5-week-old spinal cord was examined to determine if developmental changes in oligodendroglial Ca²⁺ regulation occurred in vivo. The results of these experiments indicate that the percentage of oligodendroglia responding to either ATP or carbachol markedly decreased as a function of the age of the animal used to prepare the cultures; this was not the case for the stimulation of Ca²⁺_i by histamine. The decreased sensitivity of oligodendroglia isolated from older animals could not be reversed through the addition of dibutyryl cyclic AMP. Overall, the results of these experiments indicate that developmental processes selectively influence the sensitivity of oligodendroglia to specific neuroligands and suggest that oligodendroglial processes unrelated to myelin formation may be regulated by neuroligands in vivo.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Cannabinoid effects on adenylate cyclase and phosphodiesterase activities of mouse brain.

The experiments presented in this paper examine the mechanisms underlying the ability of cannabinoids to alter the in vivo levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in mouse brain. It was found that changes in cyclic AMP levels are a composite result of direct actions of cannabinoids on adenylate cyclase (EC 4.6.1.1) activity and indirect actions involving the potentiation or inhibition of biogenic amine induced activity of adenylate cyclase. Furthermore, the long-term intraperitoneal administration of 1-(--)-delta-tetrahydrocannabinol to mice produced a form of phosphodiesterase (EC 3.1.4.17) in the brain whose activity is not stimulated by Ca2+, although its basal specific activity is similar to that of control animals. In vitro, the presence of the cannabinoids caused no significant changes in activity of brain PDE at the concentrations tested. Some correlations are presented which imply that many of the observed behavioral and physiological actions of the cannabinoids in mammalian organisms may be mediated via cyclic AMP mechanisms.     

Evolution of 3-hydroxy-3-methylglutaryl-CoA reductase and microsomal membrane fluidity throughout chick embryo development

The pattern of chick liver and brain 3-hydroxy-3-methylglutaryl-CoA reductase and its relationship with changes in microsomal membrane fluidity was studied during embryonic and postnatal development. A peak of brain activity was found at 19 days of embryonic development, while liver activity only increased after hatching. A significant increase in cholesterol content of brain microsomes occurred at about 14 days of incubation, decreasing afterwards. No significant variations were observed in liver microsomes during the same period. A similar profile was found in the phospholipid content of both brain and liver microsomes. The cholesterol/lipidic phosphorus molar ratio of brain and liver microsomes did not exhibit significant changes throughout embryonic and postnatal development. These results demonstrate that membrane-mediated control does not regulate the evolution of reductase activity during this developmental period.     

Effect of organotin compounds and hexachlorophene on brain adenosine cyclic 3',5'-monophosphate metabolism.

The effect of triethyltin (TET), triphenyltin (TPT), hexachlorophene (HCP) and cuprizone on adenosine cyclic 3',5'-monophosphate (cyclic AMP) production in rat brain was examined both in vitro and in vivo. TET and TPT inhibited basal adenylate cyclase activity of brain homogenate at a concentration as low as 1 microM in vitro but these compounds had no effect on norepinephrine (NE) and dopamine(DA)-stimluated enzyme activity. HCP and cuprizone failed to inhibit adenylate cyclase activity. In vivo TET given intravenously at a dose rate of 10 mg/kg decreased the cyclic AMP content of cerebrum, but not of medulla. TPT and HCP give intravenously and intraperitoneally respectively failed to decrease the cyclic AMP content of the cerebrum. In the case of TET the reduction in cyclic AMP content of the cerebrum was prevented by maintaining the rats normothermic after treatment. On the basis of these results the inhibition of adenylate cyclase produced by TET in brain homogenates in vitro would not appear to be involved in the development of nervous changes associated with acute TET toxicity, or in the production of progressive brain oedema caused by TET, HCP and cuprizone.     

Ontogenetic changes in levels of phosphodiesterase for adenosine 3':5'-monophosphate and glucosine 3':5'-monophosphate in the lung, brain and heart from guinea pigs.

Changes in tissue levels of the low Km phosphodiesterase for adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclc GMP) in the lung, liver, heart and brain from developing guinea pigs were studied. It was found that the contents of the soluble (cytosol) phosphodiesterase for both cyclic AMP and cyclic GMP were higher in the lung from the fetus than from the neonate and adult. The ontogenetic changes seen in the liver were qualitatively similar to thos in the lung with respect to cyclic GMP hydrolysis, while a reversed pattern of change was noted in the brain. The level of cyclic AMP phosphodiesterase was highest in the fetal heart. Throughout the fetal stage, the levels of the enzyme for cyclic GMP hydrolysis were higher than those for cyclic AMP in the lung. At or around birth, a reversal in the relative levels of the two enzymes took place; two days after birth, the level of the enzyme for cyclic AMP was 2-3times higher than thos for cyclic GMP. Kinetic analysis showed that phohphodiesterases from extracts of the lung from all developmental stages of guinea pigs had the same Km (2.6 muM) for cyclic AMP and the same Km (6.6 muM) for cyclic GMP. The relative values of V, based on assays using the same amount of enzyme protein, in decreasing order, were fetus greater than neonate greater than adult. The present findings suggest that metabolism of the two cyclic nucleotides may be closely related to developmental processes of the tissues. Moreover, the actions involving cyclic GMP may be more predominent in the fetal lung and adult brain.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Development of hormonal regulation of ion transport in embryonic chick red cells

We have found that cation transport in red cells from chick embryos is stimulated by the hormone epinephrine and that this response develops as the embryonic definitive cells mature. Sodium efflux and potassium influx are significantly stimulated (50%) by epinephrine in red cells from embryos incubated ten days or longer, whereas cation fluxes in erythroid cells from 8- or 9-day embryos are stimulated little or not at all. The effect of epinephrine may be mediated by cyclic AMP as adenylate cyclase activity in membranes isolated from embryonic red cells is only slightly stimulated at nine days, but the response increases as the cells mature to a maximum of about 180%. Also the stimulation of cation transport by epinephrine is blocked by propranolol, but not by phentolamine. Although the younger cells respond poorly to epinephrine, cyclic AMP significantly stimulates transport. The enhancement of cation fluxes by epinephrine or cyclic AMP occurs even in the presence of ouabain. Since both K influx and Na efflux are enhanced by these agents, their action is most likely on some form of the “Na-K” pump which is not ouabain sensitive resulting in a significant increase in the maximum velocity of the pump. We suggest the hypothesis that there are two classes of “Na-K” pump in these embryonic cells. One pump is similar to that found in many erythrocytes including mammalian cells in that it selectively pumps potassium in and sodium out, is ouabain-sensitive, and is primarily involved in maintaining intracellular cation concentrations. The second pump is enhanced by epinephrine via cyclic AMP, is not inhibited by ouabain, and may have lower ion selectivity. This hormone sensitive pump activity is lost as the cells mature, a process which is completed when the animal is fully grown and no longer has significant numbers of embryonic cells in its circulation.     

ACTIVITY OF CYCLIC AMP-DEPENDENT MICROSOMAL PROTEIN KINASE AND PHOSPHORYLATION OF RIBOSOMAL PROTEIN IN RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— Microsomes from rat brain exhibited protein kinase activity which was stimulated by cyclic AMP when assayed in the presence of exogenous protein substrate, such as thymus histone. In the absence of exogenous substrate some phosphorylation of microsomal protein occurred, but no stimulation by cyclic AMP could be discerned, probably because of limitations of substrate. The maximal activity of microsomal protein kinase observed in the presence of saturating concentrations of histone and the optimal concentration (5 μ m ) of cyclic AMP remained essentially unchanged from birth to early adulthood, but the magnitude of the stimulation by cyclic AMP was significantly higher at birth than at 30 days of age. Brain ribosomal proteins could be phosphorylated by the cyclic AMP-dependent brain protein kinase. Their total capacity for acceptance of phosphate by means of this phosphorylation reaction remained unchanged throughout the postnatal development of the brain. Our results are consistent with the possibility that phosphorylation of ribosomal protein mediated by cyclic AMP-dependent protein kinase may play a a role in the postnatal regulation of cerebral protein synthesis, as a result of the changes in the levels of cyclic AMP known to occur in brain during postnatal maturation.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.     

Role of cyclic AMP and related enzymes in rat lung growth and development.

Cyclic AMP levels in rat lungs showed phasic elevations which peaked during fetal, neonatal and late postnatal periods of development. Lung phospholipids showed major alterations in their levels during fetal and early neonatal life. Alterations in glycogen levels were accompanied by parallel changes in phosphorylase a/total phosphorylase activity which may be related to changes in cyclic AMP during development. Cyclic AMP levels were dependent on the relative activities of adenylate cyclase and cyclic AMP phosphodiesterase which also changed with age. Activation of adenylate cyclase by norepinephrine and NaF, and of cyclic AMP phosphodiesterase by calcium, was maximum neonatally and declined variably thereafter. These data suggest a relationship between cyclic AMP, glycogen and phospholipids during rat lung development.     

Activities and some properties of adenylate cyclase and phosphodiesterase in muscle, liver and nervous tissues from vertebrates and invertebrates in relation to the control of the concentration of adenosine 3'':5''-cyclic monophosphate.

1. The basal and fluoride-stimulated activities of adenylate cyclase, and the maximal activities of 3':5'-cyclic AMP phosphodiesterase and 3':5'-cyclic GMP phosphodiesterase, together with the Km values for their respective substrates, were measured in muscle, liver and nervous tissues from a large range of animals to provide information on the mechanism of control of cyclic AMP concentrations in these tissues. High activities of adenylate cyclase and cyclic AMP diesterase are found in nervous tissues and in the more aerobic muscles (e.g. insect flight muscles, cardiac muscle and some vertebrate skeletal muscles). The activities of these enzymes in liver are similar to those in the heart of the same animal. The Km values for the enzymes from different tissues and animals are remarkably similar. 2. The comparison of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities suggests that in vertebrate tissues only one enzyme (the high-Km enzyme), which possesses dual specificity, exists, whereas in invertebrate tissues there are at least two phosphodiesterases with separate specificities. 3. A simple quantitative model to explain the control of the steady-state concentrations of cyclic AMP is proposed. The maximum increase in cyclic AMP concentration predicted by comparison of basal with fluoride-stimulated activities of adenylate cyclase is compared with the maximum increases in concentration produced in the intact tissue by hormonal stimulation: reasonable agreement is obtained. The model is also used to predict the actual concentrations and the rates of turnover of cyclic AMP in different tissues and, where possible, these values are compared with reported values. Reasonable agreement is found between predicted and reported values. The possible physiological significances of different rates of turnover of cyclic AMP and the different ratios of high- and low-Km phosphodiesterases in different tissues are discussed.     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium

Embryonic chick (7–9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionicf strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7–9-day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio in newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanined by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium.

Embryonic chick (7-9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionic strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7--9 day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio of newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanied by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Regulation of adenosine 3':5'-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 microM) and forskolin (20 microM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 microM) while isoproterenol at 100 microM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4-6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60-70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.     

Studies on sex-organ development. The hormonal regulation of steroidogenesis and adenosine 3'':5''-cyclic monophosphate in embryonic-chick ovary.

1. We investigated the production of steroid hormones by the ovaries of the developing embryonic chick under conditions of organ culture. Radioimmunoassay techniques were used to measure the amount of steroid hormone released into the culture medium. Stimulation of the production of steroid hormones by choriogonadotropin from the urine of pregnant human was dose-dependent. Oestradio and testosterone production was optimal when 20 i.u. of gonadotropic hormone was present in the culture medium 2. During development, both left and right ovaries responded to gonadotropic hormone stimulation with a 2.5-3-fold increase in oestrogen production. However, the right ovary was twice as efficient in testosterone production as the left one. The presence of dibutyryl cyclic AMP in the culture medium of the embryonic ovaries mimicked the effect of the gonadotropic hormone. 3. The human choriogonadotropic hormone stimulated cyclic AMP production in the embryonic ovarian tissue. Thyrotropin, growth hormone and insulin had no stimulating effect. 3-Isobutyl-1-methylxanthine potentiated the gonadotropic hormone effect by increasing the concentration of cyclic AMP in the ovarian tissue. 4. The amount of cyclic AMP synthesized in the embryonic ovary was gradually increased (from 1.2 to 6.5 pmol/mg of tissue) when incubated with increasing doses of human choriogonadotropic hormone in vitro. The newly synthesized cyclic AMP reached the maximum concnentration after 30 min of incubation, then decreased at 2 h of incubation. A portion of the newly synthesized cyclic AMP was released into the culture medium. 5. At various developmental stages, both left and right embryonic-chick ovaries responded to stimulation by gonadotropic hormone with an increase in cyclic AMP production. The cyclic AMP concentration in the right ovary was 80% higher than that in the corresponding left ovary.     

Diminished Noradrenergic Stimulation Reduces the Activity of Rolipram-Sensitive, High-Affinity Cyclic AMP Phosphodiesterase in Rat Cerebral Cortex

Abstract: The present study examined the in vivo regulation of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of cyclic AMP, formed by stimulation of β-adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of cyclic AMP formed under these conditions was inhibited by the PDE4-selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6-hydroxydopamine (6-OHDA; 200 µg) decreased the rate constant of cyclic AMP hydrolysis and increased the cyclic AMP half-life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6-OHDA infusion, respectively. This was followed by the development of supersensitivity of β-adrenergic receptor-linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6-OHDA (300 µg) was used, the reduction in the rate constant of cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6-OHDA treatment, continuous blockade of β-adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of β-adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor-effector system in the brain.     

MODIFICATIONS IN SOLUBLE PROTEIN KINASE AND CYCLIC-AMP BINDING CAPACITY OF DEVELOPING RAT BRAIN

Histone and casein phosphoprotein-kinase activities were determined in rat brain soluble fraction at various stages of development. Cyclic AMP -independent or basal histone kinase activity increased, whereas cyclic AMP -dependent activity decreased in whole soluble fraction with the age. On the contrary, whole soluble cyclic AMP -dependent and -independent casein kinases activities did not show any difference during development. The percentage of activation by cyclic AMP of histone kinase activity and [³H] cyclic- AMP binding activity in the soluble fraction decreased markedly during development. By DEAE-cellulose chromatography the histone kinase was separated mainly into 4 peaks; the fourth peak was strongly stimulated by cyclic AMP . Stimulation by cyclic AMP was higher in the 4-day-old rat brains than in the 9- and 30-day-old. In the 9-day-old rats the ratio of cyclic AMP -dependent histone kinase in respect to the cyclic AMP -independent enzyme was higher than in 4- and 30-day-old rats. Casein kinase activities in the brains of 9- and 30-day-old rats were separated by DEAE-cellulose chromatography into three peaks of which the third one was stimulated by cyclic AMP . Little, if any, difference was observed for casein kinase during the development. These results suggest that brain histone and casein kinase are different enzymes: