Effects of Chronic Lithium Treatment on Agonist-Enhanced Extracellular Concentrations of Cyclic AMP in the Dorsal Hippocampus of Freely Moving Rats

Abstract: Studies on brain slices and homogenates suggest that chronic lithium treatment affects the activity of adenylate cyclases in the brain. To investigate whether chronic lithium administration influences the cyclic AMP (cAMP) synthesis in vivo, we have used microdialysis to assess lithium-induced alterations in extracellular concentrations of cAMP in the dorsal hippocampus of freely moving rats. Local infusion of noradrenaline or forskolin through the microdialysis probes produced rapid increases in the extracellular concentrations of cAMP in the dorsal hippocampus. Lithium administration for 4 weeks (serum lithium concentration of 0.8 ± 0.11 mmol/L) did not affect the baseline levels of cAMP. However, in rats fed a lithium-supplemented diet, noradrenaline- and forskolin-induced enhancement of cAMP levels was decreased in the dorsal hippocampus. The rats were videotaped 18 min before and 27 min after initiating the introduction of noradrenaline and forskolin into the dorsal hippocampus. The infusion of agonists induced a moderate behavioral excitation. Rats treated with lithium were less active compared with the control rats. Taken together, these data confirm that chronic lithium administration affects the cAMP signaling system in the brain of living animals, presumably by interfering with a site beyond the receptor level.     

Effect of certain agents on subcellular cAMP level in different areas of rat brain

The influence was studied in vitro of certain agents (adenosine, ADP, ATP, theophylline, together with F- ions) on the cAMP concentrations in the nuclear (N) and mitochondrial (M) fractions from different areas of rat brain. F- ions caused a slight decrease of the cAMP concentrations in nuclear fractions of the thalamus with hypothalamus and a marked decrease of this cyclic nucleotide in M fractions from the cerebral cortex. After incubation with adenosine and F- ions a distinct decrease of cAMP level was observed in N fractions from the midbrain and thalamus with hypothalamus and in mitochondrial fractions obtained from all the investigated regions. The incubation with ATP and F- ions resulted in a distinct decrease of cAMP values in the nuclear fractions from all regions. The concentrations of cAMP in the mitochondria of the midbrain and thalamus with hypothalamus incubated with ATP and F- ions increased 2-3 times. The incubation of the nuclear fraction with theophylline and F- ions caused an increase of cAMP concentration in the cortex and a decrease of cAMP values in the midbrain. The level of cAMP after the incubation with theophylline and fluoride on the mitochondrial fraction is increased in the cortex and decreased in the thalamus with hypothalamus.     

Long-term regulation of serotonergic activity in the rat brain via activation of protein kinase A.

Serotonergic neurons located at the base of the mammalian brain innervate practically every region of the brain and the spinal cord. These neurons exhibit spontaneous electrical discharges in a rhythmical way. Their firing frequency is modulated by serotonin autoreceptors which also regulate intracellular cAMP levels. We have investigated how elevated levels of cAMP alter the development and the functional properties of serotonergic neurons in culture. To study the influence of cAMP on the expression of genes underlying serotonergic activity, a quantitative RT-PCR approach using internal standards was developed. Cultures of embryonic rat brain serotonergic neurons were continuously treated with cAMP analogues. Increased cAMP levels had three effects. First, the neuronal morphology was changed towards that typical for mature serotonergic neurons. Second, the expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin production, was increased in dibutyryl-cAMP treated cultures. Third, the expression of the inhibitory autoreceptor (5-HT1A) was down-regulated. These results suggest the existence of a mechanism by which the neurons react to synaptic input regulating intracellular cAMP levels. Increased cAMP concentrations affect the development and cause a prolonged activation of serotonergic transmission. Since 5-HT1A receptors inhibit cAMP formation, their down-regulation argues against a negative feedback control in this system, consistent with observations in vivo.     

Cyclic adenosine-3',5'-monophosphate content in the limbic system structures of the rat brain on the administration of corticosteroids

The effect of hydrocortisone and DOCA on the cAMP content in the hypothalamus, hippocampus and striate body of the rat brain was investigated. Single (determined after 1 and 24 hours) and repeated (7 days) hydrocortison administration in a dose of 5 mg/100 g body weight was accompanied by an increase in the cAMP concentration in the brain structures under study. Single administration of DOCA in a dose of 0.5 mg/100 g body weight did not produce any changes in the cAMP level in the structures of the rat brain limbic system; however, the dose of 2.5 mg raised the cAMP level. Prolonged administration of the hormone in the above doses dod not change the cAMP level in the brain structures. Only the hippocampus showed a 210% increase in the cAMP level during DOCA administration in a dose of 0.5 mg.     

Effects of cyclic nucleotides on ornithine decarboxylase activity in mammary gland explants from mid-pregnant mice

Dibutyryl cAMP and prolactin stimulated ornithine decarboxylase activity in mouse mammary gland explants which had been preincubated with insulin and cortisol for 1 day; maximally stimulatory concentrations of dibutyryl cAMP and prolactin produced a response which was greater than the sum of the responses of prolactin and dibutyryl cAMP when tested alone. 8-Bromo-cGMP inhibited ornithine decarboxylase activity whereas other derivatives of cyclic nucleotides were without effect. Cortisol concentrations were found to be important for optimizing the dibutyryl cAMP and prolactin responses. Optimal prolactin responses were obtained with cortisol concentrations greater than 10(-7) M, whereas optimal dibutyryl cAMP responses were observed with cortisol concentrations less than 10(-7) M. Despite the differing optimal cortisol concentrations for the prolactin and dibutyryl cAMP responses, it is concluded that prolactin and dibutyryl cAMP probably stimulate ornithine decarboxylase activity in the mammary gland via the same mechanism.     

A biphasic and brain-region selective down-regulation of cyclic adenosine monophosphate concentrations supports object recognition in the rat

Background

We aimed to further understand the relationship between cAMP concentration and mnesic performance.

Methods and Findings

Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h).

Conclusions

Our results strongly suggest that a “pre-sample” early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay.     

Aspects of the biochemical toxicology of cadmium.

Cadmium, in addition to producing a variety of toxic manifestations, is known to accumulate in certain "target" organs which include liver and kidney where histological and functional damage becomes apparent. The daily intraperitoneal injection of cadmium chloride for 21 or 45 days stimulated the activities of hepatic pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1, 6-diphosphatase and glucose-6-phosphatase elevated blood glucose and urea, and lowered hepatic glycogen in rats. Whereas chronic Cd treatment failed to alter adenosine-3', 5'-monophosphate phosphodiesterase (PDE) activity, cyclic AMP (cAMY and the activity of basal and fluoride-stimulated forms of hepatic adenylate cyclase (AC) were markedly increased. However, the cAMP binding to hepatic protein kinase was decreased as was the kinase activity ration. An acute dose of Cd decreased hepatic glycogen content and increased blood glucose, serum urea, and hepatic cAMP. Chronic exposure to Cd induced adrenal hypertrophy and augmented adrenal norepinephrine and epinephrine as well as the activity of adrenal tyrosine hydroxylase. This treatment decreased prostatic and testicular weights of mature rats. Although cAMP as well as AC activity of the prostate gland were reduced, cAMP binding to the prostatic protein kinase was increased as was the activity of the cAMP-dependent form of the enzyme. Testicular AC and PDE activities, however, were stimulated, although cAMP remained unaffected. Whereas the activities of the cAMP-dependent and the independent forms of testicular protein kinase were significantly depressed, the binding of cAMP to protein kinase from testes of Cd-treated rats was not affected. In most cases, the observed metabolic alterations persisted up to 28 days on cessation of Cd administration. Subacute Cd treatment suppressed pancreatic function as evidenced by lowered serum immunoreactive insulin (IRI) in presence of hyperglycemia, as well as by partial inhibition of phentolamine-stimulated increases in serum IRI. Although chronic Cd treatment failed to alter the concentration of brain stem norepinephrine and cerebrocortical acetylcholine esterase activity, serotonin levels of brain stem were depressed and the concentration of striatal dopamine and cerebrocortical acetylcholine were significantly elevated when compared with the values seen in control nonexposed animals.     

Effect of benzimidazole derivatives and levamisole (phenylimidothiazole) on cyclic 3':5'-AMP phosphodiesterase from pig brain

The effects of phenylimidothiazole and some benzimidazole derivatives on phosphodiesterase of cyclic 3':5'-AMP (cAMP) from pig brain were studied. Depending on their concentrations the compounds tested activated or inhibited the enzyme activity. A correlation between the effects of benzimidazoles and phenylimidothiazole which is known to exert an immunomodulating action was observed.     

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...     

Enkephalin and cyclic nucleotide content in the brain structures of rats at different stages of the formation and development of alcoholic dependence

Rats with increased alcohol motivation have been found to have a rise in enkephalin levels in limbic cortex and a decrease in met-enkephalin levels in the brain basal ganglia. Reduction of met-enkephalin to leu-enkephalin ratio in basal ganglia, limbic cortex and hypothalamus may serve as an index of increased inclination to ethanol in these animals. Alcohol dependence is characterized by reduced cAMP content in the majority of brain structures studied, sharply decreased met-enkephalin levels in limbic cortex and hypothalamus, and diminished cAMP and cGMP content in hypothalamus. In the third stage of experimental alcoholism the partial normalization of met-enkephalin and cAMP levels is observed in brain structures, with cGMP content increased in hypothalamus and considerably reduced in basal ganglia.     

Activation of adenosine 3',5'-monophosphate-dependent protein kinase and its relationship to cyclic AMP and lipolysis in hamster adipose tissue

The interrelationships among cAMP-dependent protein kinase activity, lipolysis, and cellular concentrations of cAMP were investigated in hamster epididymal adipose tissue. Isoproterenol, norepinephrine, and theophylline increased the protein kinase activity assayed in tissue extracts with no added cAMP, but not in the presence of added cyclic nucleotide. The maximum rate of lipolysis was associated with a nearly three-fold increase in cAMP levels and a protein kinase activity ratio of 0.8 (the ratio of activity assayed without cAMP to that assayed with cAMP). Rates of lipolysis less than maximum were associated with lesser degrees of protein kinase activity and lower levels of cAMP. The relatively pure alpha-adrenergic agent phenylephrine partially suppressed the isoproterenol-stimulated protein kinase activity, lipolysis, and cAMP levels. Conversely, the alpha-adrenergic blocking agent phentolamine increased the activity of protein kinase and cAMP levels in adipose tissues exposed to norepinephrine. These data are consistent with the primary role for cAMP and its dependent protein kinase in control of lipolysis in adipose tissue. Moreover, our data are consistent with the view that the antilipolytic action of alpha-adrenergic agents is mediated by a decrease in activity of protein kinase, caused by a decrease in cellular cAMP concentrations.     

Prostacyclin (PGI2) induces rapid depletion of cyclic AMP levels in brain nuclei of rats

Intravenous injection of prostacyclin (100 micrograms/kg) in rats resulted in a decrease of systolic blood pressure within 2 minutes. Concentrations of cAMP in 15 brain regions and nuclei were determined by radioimmunoassay. In lower brain stem nuclei, such as the nucleus of the solitary tract and the lateral reticular nucleus (A1 and C1 catecholaminergic cell groups) cAMP levels were depleted significantly, while in others, including the locus coeruleus and the periaqueductal central gray, cAMP levels did not show any alterations. Levels of cAMP were also depleted in some of the hypothalamic nuclei (periventricular, anterior hypothalamic, ventromedial), and in cerebral cortical areas. Lowered cAMP levels in brain areas might indicate lower cellular activity in cells participating in baroreceptor control mechanisms.     

Activation of protein kinase in thyroid slices by thyroid-stimulating hormone.

Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone-induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G-100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NcCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [3H]cAMP binding. These results are compatible with the concept that the TSH-mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.     

Inactive forms of the catalytic subunit of protein kinase A are expressed in the brain of higher primates

It is well documented that the beta-gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N-terminal end encoded by alternative use of several exons located 5' to exon 2 in the human, bovine and mouse Cbeta gene. In the present study, we demonstrate the expression of six novel human Cbeta mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbetaDelta4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbetaDelta4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbetaDelta4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP-responsive element binding protein. Furthermore, co-expression of CbetaDelta4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CbetaDelta4 variants associate with both RI and RII in a cAMP-independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CbetaDelta4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.     

Mutants of PC12 cells with altered cyclic AMP responses.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.     

Metabolism of cyclic AMP in isolated renal tubules: effects of prostaglandins and parathyroid hormone.

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) with maximal effects of PGE1 being 6-8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2-3 fold greater in tubules exposed to PTH than to PGE1. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates. The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Calcium and cyclic AMP involvement in the regulation of juvenile hormone biosynthesis in Diploptera punctata

We have examined the effects of brain extracts on cAMP levels in the CA as well as their effect on JH biosynthesis in vitro. Brain extracts induced a dose-dependent elevation in intracellular cAMP levels (as measured by RIA) within 10 min of treatment. Significant (P < 0.05) elevations in cAMP were observed with concentrations of extract as low as 0.01 brain equivalents. Maximal elevation (120–140% over controls) was obtained with 0.25 brain equivalents and greater. Elevated extracellular calcium (10 mM) substantially reduced the inhibitory effects of brain extracts on JH biosynthesis in vitro when compared to normal controls at 1.3 mM calcium. High calcium had relatively little effect on basal cAMP levels in the CA. In contrast, the use of calcium-free medium with EGTA caused a dramatic increase in cAMP levels. High calcium did not affect the ability of the brain extracts or the ability of the adenylate cyclase activator forskolin to elevate cAMP levels. These results provide further evidence that cAMP acts as a second messenger for a putative allatostatin from the brain. As well, these results further suggest that both calcium-dependent and calcium-independent (involving cAMP) pathways function to reduce JH biosynthesis in the CA of D. punctata.     

Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.     

Brain and ring gland cyclic AMP and cyclic GMP levels during initiation and termination of pupal diapause in flesh flies

Brain and ring gland concentrations of cyclic AMP were much higher shortly after pupariation in long day (non-diapause destined) flesh flies than in short day (destined for pupal diapause) flies. This difference was most striking in the ring gland (6 times higher in long day flies). Cholera toxin elevated brain-ring gland cAMP four-fold, thus accounting for its efficacy in averting diapause. No differences in cyclic GMP levels were detected between long and short day flies at pupariation. At diapause termination cAMP and cGMP concentrations in the brain and ring gland and cAMP in whole body homogenates changed only slightly, but whole body concentrations of cGMP rose markedly.     

Metabolic Changes in Cerebral Cortex, Hippocampus, and Cerebellum During Sustained Bicuculline-Induced Seizures

Abstract— The objective of the present experiments was to study metabolic correlates to the localization of neuronal lesions during sustained seizures. To that end, status epilepticus was induced by i.v. administration of bicuculline in immobilized and artificially ventilated rats, since this model is known to cause neuronal cell damage in cerebral cortex and hippocampus but not in the cerebellum. After 20 or 120 min of continuous seizure activity, brain tissue was frozen in situ through the skull bone, and samples of cerebral cortex, hippocampus, and cerebellum were collected for analysis of glycolytic metabolites, phosphocreatine (PCr), ATP, ADP, AMP, and cyclic nucleotides. After 20 min of seizure activity, the two “vulnerable” structures (cerebral cortex and hippocampus) and the “resistant” one (cerebellum) showed similar changes in cerebral metabolic state, characterized by decreased tissue concentrations of PCr, ATP, and glycogen, and increased lactate concentrations and lactate/ pyruvate ratios. In all structures, though, the adenylate energy charge remained close to control. At the end of a 2-h period of status epilepticus, a clear deterioration of the energy state was observed in the cerebral cortex and the hippocampus, but not in the cerebellum. The reduction in adenylate energy charge in the cortex and hippocampus was associated with a seemingly paradoxical decrease in tissue lactate levels and with failure of glycogen resynthesis (cerebral cortex). Experiments with infusion of glucose during the second hour of a 2-h period of status epilepticus verified that the deterioration of tissue energy state was partly due to reduced substrate supply; however, even in animals with adequate tissue glucose concentrations, the energy charge of the two structures was significantly lowered. The cyclic nucleotides (cAMP and cGMP) behaved differently. Thus, whereas cAMP concentrations were either close to control (hippocampus and cerebellum) or moderately increased (cerebral cortex), the cGMP concentrations remained markedly elevated throughout the seizure period, the largest change being observed in the cerebellum. It is concluded that although the localization of neuronal damage and perturbation of cerebral energy state seem to correlate, the results cannot be taken as. evidence that cellular energy failure is the cause of the damage. Thus, it appears equally probable that the pathologically enhanced neuronal activity (and metabolic rate) underlies both the cell damage and the perturbed metabolic state. The observed changes in cyclic nucleotides do not appear to bear a causal relationship to the mechanisms of damage.