Inhibition of 3',5'-nucleotide phosphodiesterase in guinea pig cerebral cortical slices

Several compounds have been tested for their activity as inhibitors of 3′,5′-nucleotide phosphodiesterase in brain cortical slices from guinea pig. SQ 20,009 (1-ethyl-4-isopropylidenehydrazino)-1H-pyrazolo (3,4-b)pyridine-5-carboxylate, ethylester, hydrochloride), a very potent inhibitor of 3′,5′-nucleotide phosphodiesterase from rat and rabbit brain shows only moderate activity as 3′,5′-nucleotide phosphodiesterase inhibitor when tested in brain slices. It enhances cyclic AMP accumulation only when slices are stimulated by histamine. It does not affect cyclic AMP levels when histamine/norepinephrine are used as stimuli of cyclic AMP formation and decreases the activity of adenosine as stimulant slightly. Ro 20–1724 (4-(3-butoxy-4-methoxy)-2-imidazolidinone) a potent inhibitor of canine cerebral cortex PDE activity effectively augments the increase in cyclic AMP under all stimulating conditions mentioned, as does to a somewhat smaller extent the more water soluble Ro 20–2926 (4-(3-ethoxy-ethoxy-4-methoxy)-2-imidazolidinone). Dose-response curves for Ro 20–1724 under three stimulating conditions of increased cyclic AMP formation (0.1 mm histamine, 0.1 mm histamine/0.1 mm norepinephrine, 0.1 mm adenosine) yield an ED₅₀ of about 20 μm in all instances. A significant increase over respective controls is seen even at 1 μm Ro 20–1724 (histamine/norepinephrine). The drugs may be useful as tools for studying the regulation of cyclic AMP levels in the central nervous system.     

Cysteine Sulfinic Acid in the Central Nervous System: Antagonistic Effect of Taurine on Cysteine Sulfinic Acid-Stimulated Formation of Cyclic AMP in Guinea Pig Hippocampal Slices

The stimulatory effect of cysteine sulfinic acid on cyclic AMP formation was examined in slices from three different regions of guinea pig brain. The inhibitory effect of taurine on the stimulated formation of cyclic AMP was also studied. Cysteine sulfinic acid (1--10 mM) greatly increased the cyclic AMP level in striatal, cortical, and especially hippocampal slices. In hippocampal slices, taurine (0.1--30 mM) markedly lowered the increase of cyclic AMP induced by cysteine sulfinic acid, but not that induced by glutamate or aspartate. In this region, taurine also reduced the stimulatory effects on cyclic AMP formation of adenosine, norepinephrine, and histamine, but not of depolarizing agents. It did not, however, inhibit the effects of any of these stimulants in cortical slices. These results suggest that sulfur-containing amino acids, such as cysteine sulfinic acid and taurine, regulate the cyclic AMP level in the hippocampus.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

Ecto-5''-Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.     

Inhibition of Cyclic AMP Formation by a Selective Metabotropic Glutamate Receptor Agonist

It is well documented that the effects of excitatory amino acid (EAA) agonists on phosphoinositide hydrolysis involve a GTP-binding protein-linked or "metabotropic" receptor mechanism. The mechanisms by which EAAs alter cyclic AMP levels in brain slices, however, are not yet clear. In this study, the selective metabotropic EAA agonist trans-(+-)-1-aminocyclopentane-1,3-dicarboxylic acid and its isomers were examined for effects on basal and forskolin-stimulated cyclic AMP formation in slices of the rat hippocampus. Trans-(+-)-1-Aminocyclopentane-1,3-dicarboxylic acid had little effect on basal cyclic AMP but inhibited forskolin-stimulated cyclic AMP formation in a biphasic manner. The 1S,3R isomer of 1-aminocyclopentane-1,3-dicarboxylic acid produced potent but only partial (approximately 50%) inhibition of forskolin-stimulated cyclic AMP formation. 1R,3S-1-Aminocyclopentane-1,3-dicarboxylic acid fully inhibited forskolin-stimulated cyclic AMP but with lower potency than the 1S,3R isomer. These results show that in addition to the formation of phosphoinositide-derived second messengers, the cellular consequences of selectively activating hippocampal metabotropic EAA receptors include an alteration of cellular cyclic AMP levels.     

CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: POSSIBLE REGULATION OF PHOSPHODIESTERASE ACTIVITY BY CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE AND CALCIUM IONS

Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) accumulates in guinea pig cerebral cortical slices during incubation with histamine, histamine + noradrenaline and adenosine. Noradrenaline does not enhance cyclic AMP formation. In the absence of Ca²⁺ ions and presence of 1 mM-EGTA in the Krebs-Ringer bicarbonate medium the effects of histamine, histamine + noradrenaline and adenosine are significantly enhanced and noradrenaline elicits an increase in cyclic AMP over control levels. When histamine is used as stimulant, cyclic AMP levels start to decline after only 5 min. However, in the absence of calcium and in the presence of EGTA in the medium this decline is not observed and cyclic AMP levels continue to rise for a considerable period of time. In normal medium, responses to restimulation by histamine or histamine + noradrenaline are greatly reduced in magnitude after a prior stimulation by these putative neurotransmitters. In contrast, when calcium is omitted from the incubation medium and 1 mM-EGTA is included, cyclic AMP levels increase to normal values at a second stimulation with histamine or histamine + noradrenaline. When slices are preincubated for various periods of time with histamine before addition of noradrenaline, the accumulation of cyclic AMP is significantly reduced as compared to levels obtained when histamine + noradrenaline were added simultanously. This decline in the overall response to histamine + noradrenaline is not observed when preincubation with histamine and subsequent incubations with histamine + noradrenaline are performed in Ca²⁺-free, 1 mM-EGTA containing buffer. Also preincubation with noradrenaline in normal, calcium-containing medium does not affect the total amount of cyclic AMP accumulating in the brain slices. The results are discussed in terms of an activation of phosphodiesterase within the cerebral cortical slices by increased levels of intracellular, freely available calcium which is mediated by the elevation of cyclic AMP concentration following hormonal stimulation.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

CYCLIC NUCLEOTIDES and PROTEIN KINASE ACTIVITY IN THE RAT BRAIN POSTMORTEM

Abstract— The components of the cyclic nucleotide system were studied in rat brain at various times after death to determine the stability of the system postmortem. The concentration of cyclic AMP in 4 brain regions was highest 10 min after death and declined to stable levels within 6 h after death. At this time concentrations approximated those normally seen in vitro. The concentration of cyclic GMP in brain regions fell markedly postmortem and was undetectable 6 or 16 h after death. Nor-epinephrine-stimulated cyclic AMP accumulation measured in vitro in slices of the cerebral cortex was the same 10 min and 16 h postmortem. In the cerebellum, however, accumulated levels of cyclic AMP were lower 16 h postmortem, although the degree of stimulation elicited by norepinephrine was the same 10 min and 16 h after death. Cyclic AMP-dependent and independent protein kinase activities were detected in the rat brain at all times after death. There was no change in the cyclic AMP-dependent enzyme activity postmortem, but activity in the absence of cyclic AMP was significantly higher immediately after death compared to 6 or 16 h postmortem. These studies indicate that it should be possible to study the cyclic AMP system in human brain tissue obtained at autopsy.     

INTERRELATIONSHIPS AMONG THE LEVELS OF ATP, ADENOSINE AND CYCLIC AMP IN INCUBATED SLICES OF GUINEA-PIG CEREBRAL CORTEX: EFFECTS OF DEPOLARIZING AGENTS, PSYCHOTROPIC DRUGS AND METABOLIC INHIBITORS

—Adenine nucleotides of guinea-pig cerebral cortical slices were labelled during a 40 min incubation with [¹⁴C]adenine. Subsequent incubation of cortical slices with depolarizing agents, such as veratridine, ouabain, batrachotoxin and high concentrations of potassium ions, or with certain psychotropic drugs such as chlorpromazine, chlorimipramine or prenylamine resulted in a reduction in both endogenous and radioactive ATP, accompanied by a marked increase in levels of both endogenous and radioactive cyclic AMP. Reduction of ATP levels during incubation with depolarizing agents, such as veratridine, is probably associated with increased activity of membranal Na⁺-K⁺-activated ATPase, while the reduction elicited by psychotropic drugs is proposed to be due to inhibition of mitochondrial synthesis of ATP. With both classes of compounds reduction of ATP levels results in enhanced formation and efflux of adenosine which stimulates formation of cyclic AMP from intracellular ATP in the compartments of brain slices which contain the cyclic AMP-generating systems. Certain classical metabolic inhibitors such as 2,4-dinitrophenol, azide, 1,2-naphthoquinone-8-sulfonate and cyanide also reduce ATP levels and in the case of 2,4-dinitrophenol, cyanide, and azide elicit small but significant accumulations of cyclic AMP. With certain metabolic inhibitors reduction of ATP within the cyclic AMP generating compartments would appear to prevent or reduce the accumulation of cyclic AMP elicited by amines, adenosine or veratridine.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Effects of Lithium on Inositol Phospholipid Hydrolysis and Inhibition of Dopamine D1 Receptor-Mediated Cyclic AMP Formation by Carbachol in Rat Brain Slices

The in vitro and ex vivo effects of lithium on muscarinic cholinergic inositol phospholipid hydrolysis and muscarinic cholinergic inhibition of dopamine D1-receptor-stimulated cyclic AMP formation were examined in rat brain slices. Following chronic lithium feeding, carbachol-stimulated inositol phosphate accumulation was reduced ex vivo in slices of cerebral cortex but not in striatal slices. Lithium (1 mM) in vitro had no direct effect on dopamine D1-receptor-stimulated cyclic AMP formation, but enhanced the inhibitory effect of carbachol on the D1 response, in striatal slices, and this was not significantly altered by prior lithium feeding. Lithium therefore has effects on two discrete muscarinic responses in rat brain which are apparently maintained after chronic exposure to the ion and might be relevant to its antimanic actions.     

CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA-PIG CEREBRAL CORTICAL SLICES: STUDIES ON THE ROLE OF ADENOSINE

Abstract— In guinea-pig cerebral cortical slices levels of cyclic AMP increase in response to adenosine to about 200pmol/mg protein within 10 min and stay at that level up to 30 min. In the absence of calcium ions and the presence of 1mm -EGTA in the Krebs-Ringer-bicarbonate medium the effect of adenosine is enhanced, cyclic AMP levels rise to about 600 pmol/mg protein within 30 min. In normal and calcium deficient media restimulation of cyclic AMP formation with adenosine is possible after a prior stimulation with adenosine. When slices are preincubated for various periods of time with histamine or adenosine before addition of the complementary agent i.e. adenosine or histamine cyclic AMP levels obtained are unaltered compared to levels seen when adenosine and histamine are added together. Slices which are rendered unresponsive to stimulation with histamine + noradrenaline by a prior incubation with these agents do not regain any response during a 100 min period of incubation in medium. The PDE inhibitors diazepam, SQ 66007 and isobutylmethylxanthine are capable of restoring the sensitivity of the slices to histamine + noradrenaline. This suggests an involvement of PDE in the unresponsive phase of the slices. Addition of adenosine to slices not affected by histamine + noradrenaline does reestablish the response of these slices to the neurohormones. A dose-response curve of adenosine for the interaction with histamine + noradrenaline yields an ED₅₀ of 16 μM using sensitive or desensitized slices. An adenosine concentration of only 7 μM is necessary to restore the original increase of cyclic AMP in response to histamine + noradrenaline to slices insensitive to the biogenic amines. The data are discussed in terms of a possible activation of PDE within cerebral cortical slices from guinea-pig. Adenosine may reverse this activation. The possibility of inactivation of adenylate cyclase during stimulation of cyclic AMP formation and the role of adenosine and PDE inhibitors in this process is being considered.     

Effect of Isozyme-Selective Inhibitors of Phosphodiesterase on Histamine-Stimulated Cyclic AMP Accumulation in Guinea-Pig Hippocampus

Addition of histamine (0.1 mM) to guinea-pig hippocampal slices causes a 20- to 30-fold increase in the accumulation of cyclic AMP compared with basal levels. This accumulation represents a balance between cyclic AMP production by adenylate cyclase and cyclic AMP breakdown mediated by phosphodiesterase (PDE). However, brain tissues are known to contain several different PDE isozymes. To determine which are involved in this response to histamine, the effect of isozyme-specific PDE inhibitors on cyclic AMP accumulation was examined in the hippocampus. MB 22948 (0.1 mM), an inhibitor of PDEs I and II, had no significant effect on the response to either 1 microM or 0.1 mM histamine. SKF 94120 (0.1 mM), a PDE III inhibitor, was also without effect in the presence of 1 microM histamine, although with 0.1 mM histamine, it caused a weak (1.25-fold compared with control), but statistically significant, enhancement of cyclic AMP accumulation. However, both rolipram (0.1 mM), a PDE IV inhibitor, and 3-isobutyl-1-methylxanthine (0.1 or 1 mM), an inhibitor of all forms of PDE, significantly increased cyclic AMP accumulation (2.8- to 6.5-fold compared with controls), and the relative size of this effect decreased with increasing histamine concentration. It is concluded that PDE IV is the main PDE isozyme involved in cyclic AMP turnover in guinea-pig hippocampal slices responding to histamine.     

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.     

Vasoactive Intestinal Peptide Increases Acetylcholine Synthesis by Rat Hippocampal Slices

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP) might have a presynaptic modulatory effect at cholinergic terminals in the rat hippocampal formation. The exposure of rat hippocampal slices to VIP increased [3H]acetylcholine ([3H]ACh) synthesis from the precursor [3H]choline when tissue was incubated in normal or in high K+ medium; the maximal effect was apparent at 10(-8) M VIP and 10(-7) M VIP, respectively. Also, 10(-7) M VIP increased the activity of choline acetyltransferase (ChAT) in a hippocampal homogenate system. The increased synthesis by hippocampal slices was not the result of a VIP-induced alteration in either the basal release of ACh or the uptake of choline via the high-affinity uptake system. The increase in ACh synthesis induced by VIP in hippocampal slices was not associated with either adenylate cyclase or protein kinase C second messenger systems. There was no correlation between the effect of VIP on cyclic AMP production with that on ACh synthesis; also, forskolin, an activator of adenylate cyclase that increased cyclic AMP production 3.5-fold, did not mimic the effect of VIP on ACh synthesis. Similarly, there was no effect of the protein kinase C activator, phorbol myristate acetate, on ACh synthesis in hippocampal slices. However, the effect of VIP to increase ACh synthesis was not evident in the absence of extracellular calcium, suggesting that the effect of VIP is mediated by a calcium-requiring mechanism. The results suggest that, in the rat hippocampus, VIP has a presynaptic action at cholinergic terminals that results in enhanced synthesis of ACh, possibly by an action that alters ChAT activity.     

Phorbol esters down-regulate protein kinase C in rat brain cerebral cortical slices

The effect of phorbol esters on cyclic AMP production in rat cerebral cortical slices was studied using a prelabelling technique to measure cyclic nucleotide accumulation. Cholera toxin-stimulated cyclic AMP accumulation was enhanced approximately 2-fold by phorbol 12-myristate, 13-acetate (PMA) which alone had no effect on cyclic AMP production. The augmentation by PMA was maximal within the first hour of incubation, decreasing progressively thereafter. Protein kinase C activity was decreased 80-90% during a 3 hr exposure to PMA, as was 3H-phorbol 12,13-dibutyrate binding. Both phosphatidyl serine and arachidonic acid were found to enhance protein kinase C activity in a concentration-dependent manner, an effect that was attenuated by prolonged incubation of the brain tissue with PMA. The results indicate that exposure of brain slices to phorbol esters causes a down-regulation of rat brain protein kinase C, and that this modification corresponds with a decrease in the ability of PMA to augment cyclic AMP production, suggesting a functional relationship between the two systems in rat brain.     

Activation of Metabotropic Glutamate Receptors in the Hippocampus Increases Cyclic AMP Accumulation

The selective metabotropic glutamate receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans-ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis-linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans-ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R- and 1S,3S-ACPD (but not 1R,3S-ACPD) induce a concentration-dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1S,3R-ACPD-stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.     

Calmodulin Involvement in Stress- and Corticosterone-Induced Down-Regulation of Cyclic AMP-Generating Systems in Brain

Manipulation of the hypothalamic-pituitary-adrenal axis selectively alters alpha-adrenergic potentiation of the cyclic AMP response to beta-adrenergic receptor stimulation in rat cerebral cortex. Calcium has been implicated in this alpha-receptor-mediated response, which may involve activation of phospholipases A2 and C and/or calmodulin-dependent adenylate cyclase. We therefore investigated the effects of stress and corticosterone (CORT) on membrane calmodulin-dependent adenylate cyclase and noradrenaline-stimulated cyclic AMP accumulation in brain slices. Repeated stress for 21 days selectively attenuated the adenylate cyclase response to calcium/calmodulin in cerebral cortex membranes, without affecting basal or forskolin-stimulated enzyme activity. There was no such effect in hippocampal membranes. The same pattern of response was elicited by daily CORT injection (50 mg/kg s.c.) for 21 days, while vehicle injection had no effect. CORT in the drinking water (400 micrograms/ml) elicited the same reduction of body weight as CORT injections, but had no effect on calmodulin adenylate cyclase. In parallel with calmodulin adenylate cyclase, cyclic AMP accumulation elicited by noradrenaline in slices of cerebral cortex was suppressed by both stress and daily CORT injections, with smaller effects observed with CORT in the drinking water. Unlike calmodulin adenylate cyclase, noradrenaline-stimulated cyclic AMP accumulation in hippocampus showed the same suppression as that in cerebral cortex. These results are discussed in relation to the differential mode of coupling of alpha-adrenergic receptors to cyclic AMP-generating systems between brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.