ACCUMULATION OF CYCLIC ADENOSINE 3', 5'-MONOPHOSPHATE IN CEREBRAL CORTICAL SLICES FROM RAT AND MOUSE: STIMULATORY EFFECT OF α- AND β-ADRENERGIC AGENTS AND ADENOSINE

Abstract— Norepinephrine, epinephrine, isoproterenol, and adenosine elicit enhanced accumulations of cyclic AMP in incubated slices of rat cerebral cortex. Combinations of norepinephrine, epinephrine, isoproterenol, or histamine with adenosine have a greater than additive effect on cyclic AMP levels. The effects of isoproterenol appear to be mediated via a classical β-adrenergic receptor whereas the effects of norepinephrine appear due to interactions with both α- and β-adrenergic receptors. The presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiates the effects of the catecholamines and reveals a histamine-mediated increase in cyclic AMP levels. After an initial stimulation of cyclic AMP formation with norepinephrine, followed by washing of the slices, the cyclic AMP-generating system is unresponsive to norepinephrine but does respond to an adenosine-norepinephrine combination. In mouse cerebral cortical slices, catecholamines appear to elicit an accumulation of cyclic AMP primarily via interaction with a β-adrenergic receptor.     

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.     

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.     

Characteristics of the catecholamine and histamine receptor sites mediating accumulation of cyclic adenosine 3',5'-monophosphate in guinea pig brain

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in [³H]adenosine 3′,5′-monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the [³H]-cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α-adrenergic receptor in all three areas where epinephrine was effective; α-adrenergic stimulation, but not β-adrenergic stimulation, increased levels of [³H]-cyclic AMP. Furthermore, α-, but not β-adrenergic blocking agents, prevented the epinephrine- induced increase of both [³H]- and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine-induced increase of both [³H]- and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α-adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

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.     

ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: EFFECT OF BENZODIAZEPINES

Several benzodiazepines, diazepam, chlordiazepoxide, desmethyldiazepam, methyloxazepam and oxazepam, potentiate the accumulation of cyclic AMP elicited by histamine and histamine: noradrenaline in cerebral cortical slices of guinea pig. In addition, these drugs increase basal levels of cyclic AMP by about 100 per cent. When adenosine is used to stimulate cyclic AMP formation only diazepam, desmethyldiazepam and methyloxazepam are increasing cyclic AMP levels significantly over respective controls. The order of potency is: diazepam > desmethyldiazepam > methyloxazepam > oxazepam > chlordiazepoxide. Diazepam decreases the rate of degradation of cyclic AMP after removal of the stimulatory agents (histamine : noradrenaline). Dose response curves for diazepam under two stimulatory conditions are shown. A significant effect is obtained at 50 μm -diazepam and an ED₅₀ of 40 μm is calculated with histamine as the stimulatory agent. When cyclic AMP formation is elicited by histamine : noradrenaline a significant effect of diazepam is seen at 10 μm and an ED₅₀ of 16 μm is obtained. These results lend support to the hypothesis that the psychotropic action of the benzodiazepines may, at least in part, involve the cyclic AMP generating systems of the central nervous system.     

CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE FORMATION IN GUINEA-PIG BRAIN SLICES: EFFECT OF H1- AND H2-HISTAMINERGIC AGONISTS

—A variety of histamine analogs elicit accumulations of radioactive cyclic AMP in guinea-pig neocortical and hippocampal slices labelled during a prior incubation with [¹⁴C]adenine. The H₁agonist, 2-aminoethylthiazole, elicits accumulation of cyclic AMP in neocortical and hippocampal slices both in the absence or presence of adenosine. The presence of adenosine increases the maximum response to 2-aminoethylthiazole and decreases the EC₅₀ by nearly 10-fold. In the absence of adenosine the effects of 2-aminoethylthiazole are antagonized in hippocampal slices by both d-brompheniramine and metiamide, while in the presence of adenosine only d-brompheniramine is an effective antagonist. The H₂-agonist, 4-methylhistamine, elicits a somewhat smaller accumulation of cyclic AMP than does 2-aminoethylthiazole in both cortical and hippocampal slices. In the presence of adenosine the response to 4-methylhistamine is enhanced, but is markedly lower than that seen with the combination of adenosine and 2-aminoethylthiazole. The dose-response relationship for 4-methylhistamine in the presence of adenosine appears in hippocampal slices to consist of two components. The response to 4-methylhistamine in the absence of adenosine is blocked by metiamide, while in the presence of adenosine the response is partially blocked by both H₁ and H₂-antagonists. The accumulation of cyclic AMP elicited by histamine is greatly increased by adenosine but the EC₅₀ is not significantly decreased. The results suggest that (i) both H₁- and H₂-receptors regulate cyclic AMP-formation in the central nervous system, (ii) the synergism between adenosine and histamine is mediated primarily by interaction with H₁-receptors and (iii) that adenosine greatly increases the affinity of the H₁-receptors for both H₁ and H₂-agonists without affecting its affinity for histamine.     

Accumulation of inositol phosphates and cyclic AMP in guinea-pig cerebral cortical preparations: Effects of norepinephrine,histamine, carbamylcholine and 2-chloroadenosine

Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [³H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by α₁-adrenergic, 5HT₂-serotonergic and H₂-histaminergic receptors. VIP-elicited accumulations of cyclic AMP are also augmented through stimulation of α₁-adrenergic, 5HT₂-serotonergic and H₁-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [³H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [³H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2-chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices.     

Accumulation of inositol phosphates and cyclic AMP in guinea-pig cerebral cortical preparations. Effects of norepinephrine, histamine, carbamylcholine and 2-chloroadenosine

Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [3H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by alpha 1-adrenergic, 5HT2-serotonergic and H2-histaminergic receptors. VIP-elicited accumulations of cyclic AMP are also augmented through stimulation of alpha 1-adrenergic, 5HT2-serotonergic and H1-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [3H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [3H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2-chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices.     

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.     

Depolarization-evoked accumulation of cyclic AMP in brain slices: inhibition by exogenous adenosine deaminase

In guinea pig cerebral cortical slices labeled during a prior incubation with radioactive adenine, electrical stimulation or the presence of depolarizing agents such as veratridine, ouabain, and high concentrations of K⁺ elicit a marked accumulation of radioactive cyclic AMP. This accumulation is reduced in all cases by the presence of theophylline, a compound that antagonizes the stimulatory effects of adenosine on cyclic AMP accumulation in brain slices. Exogenous adenosine deaminase also reduced the accumulation of cyclic AMP elicited by electrical stimulation, veratridine, and high concentrations of K⁺. Thus, adenosine formed in neuronal compartments under depolarizing conditions appears to be released into the extracellular medium as a prerequisite to stimulation of the cyclic AMP-generating system. Adenosine deaminase does not prevent the reduction in levels of ATP under depolarizing conditions, nor does it antagonize the accumulation of cyclic AMP elicited by a combination and norepinephrine. Adenosine deaminase does not, however, prevent the accumulations of cyclic AMP elicited by the depolarizing agent, ouabain.     

Is the Adenosine Receptor Modulation of Histamine-Induced Accumulation of Inositol Phosphates in Cerebral Cortical Slices Mediated by Effects on Calcium Ion Fluxes?

In this report, we show that under conditions designed to provide an initially uniform incorporation of [3H]inositol into mouse and guinea pig cerebral cortical slices prior to agonist stimulation, the accumulation of 3H-inositol phosphates (3H-InsPx, x = 1-4) induced by histamine in mouse and guinea pig cerebral cortical slices increased in a quasilinear manner with increasing added calcium. Raising the ambient calcium ion concentration failed to reduce the adenosine receptor-mediated inhibition of the histamine-induced 3H-InsPx response in mouse cerebral cortical slices. Similarly, the potentiation of the histamine response by adenosine receptor activation in guinea pig cerebral cortical slices was unaffected by lowering the added calcium ion concentration. The presence of the calcium ionophore A23187 (33 microM) produced 3H-InsPx responses in both mouse and guinea pig cerebral cortical slices, which were not affected by the presence of the stable adenosine analogue 2-chloroadenosine. A23187 also potentiated the accumulation of 3HInsPx induced by histamine in both species. Both the inhibitory and potentiatory modulations of the histamine response by 2-chloroadenosine in mouse and guinea pig, respectively, were still apparent in the presence of A23187. These results indicate that the histamine-induced 3H-InsPx accumulations in both mouse and guinea pig cerebral cortical slices are sensitive to variations in calcium ion concentrations. However, the adenosine receptor modulations of the histamine responses are relatively insensitive to fluctuations in either extra- or intracellular calcium ion concentrations, and thus cannot be mediated by effects on calcium ion movements.     

IN VIVO CHANGES OF CEREBRAL CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE INDUCED BY BIOGENIC AMINES: ASSOCIATION WITH PHOSPHORYLASE ACTIVATION

—The intravenous injection of adrenaline, isoprenaline and histamine to 4-6-day-old chicks resulted in a rapid increase in the cyclic AMP content of cerebral hemispheres that had been removed and frozen within 0·5 s using a freeze-blowing technique. Noradrenaline, dopamine, adenosine, 5-HT and acetylcholine did not significantly alter the nucleotide concentration in vivo. Addition of adrenaline, isoprenaline and histamine to incubated chick cerebral cortex slices also increased the cyclic AMP content of the tissue. Noradrenaline was considerably less potent than these amines and adenosine was ineffective. Low phosphorylase a levels (16 per cent of total activity) were observed in instantaneously frozen cerebral hemispheres of untreated chicks. The injection of adrenaline, isoprenaline and histamine resulted in a rapid conversion of phosphorylase b to a and a significant fall in tissue glycogen. Administration of noradrenaline was without effect on the relative forms of phosphorylase and also failed to influence cerebral glycogen. Phosphorylase activation was not observed in chick cerebral slices under conditions producing large increases in cyclic AMP. It is suggested that in vivo phosphorylase activation and subsequent glycogenolysis may occur, at least in part, in glia and that these cells may be damaged during preparation of cerebral slices. The results provide evidence of a metabolic role for cyclic AMP in cerebral tissue.     

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 incubated slices from discrete regions of squirrel monkey cerebral cortex: effect of norepinephrine, serotonin and adenosine

THE ROLE of adenosine 3′5′-monophosphate (cyclic AMP) in the central nervous system is as yet unknown. However, a variety of putative neurotransmitters such as norepinephrine, serotonin and histamine have been found to elicit enhanced accumulations of cyclic AMP in cerebral cortical slices from a variety of species (SHIMIZU and DALY, 1972; FORN and KRISHNA, 1971; SHIMIZU, TANAKA, SUZUKI and MATSUKADO, 1971; FUMAGALLI, BERNAREGGI, BERTI and TRABUCCHI, 1971). In addition, depolarization of membranes in cortical slices elicits a marked accumulation of cyclic AMP, probably mediated through the action of adenosine (KAKIUCHI, RALL and MCILWAIN, 1969; SHIMIZU. CREVELING and DALY, 1970). These results, obtained with grey matter from the entire cerebral cortex, suggest an intimate relationship between neuronal activity, release of putative neurotransmitters, and enhanced accumulation of cyclic AMP in brain tissue. Since innervation and electrical activity in the cerebral cortex differs among functionally distinct neocortical areas and in the histologically distinct limbic cortex, it appeared possible that these differences would also be manifest in the regional control of cyclic AMP levels. We now report that the response of the cyclic AMP generating system to putative neurotransmitters and adenosine does differ among discrete functional regions of the squirrel monkey cerebral cortex.     

THE EFFECT OF ELECTRICAL STIMULATION UPON THE ACCUMULATION OF ADENOSINE 3'',5''-PHOSPHATE IN ISOLATED CEREBRAL TISSUE

The application of electrical pulses to slices of guinea pig cerebral cortex led to an increase in the levels of adenosine 3′,5′-phosphate (cyclic 3′,5′-AMP) of more than 11-fold within 10 min. This effect of electrical pulses was severely reduced in the presence of theophylline. Cyclic 3′,5′-AMP accumulation in slices was increased in the presence of norepinephrine and histamine about 1·5-fold and six-fold, respectively; the effect of electrical pulses was augmented in the presence of maximal amounts of either amine. For these and other reasons, the accumulation of cyclic 3′,5′-AMP induced by electrical stimulation cannot be ascribed to the release and action of either histamine or norepinephrine.     

Cyclic AMP-Generating Systems in Cell-Free Preparations from Guinea Pig Cerebral Cortex: Loss of Adenosine and Amine Responsiveness Due to Low Levels of Endogenous Adenosine

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine-labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP-generating systems. It is proposed that, in particulate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or coupling of receptors to cyclic AMP-generating systems.     

Activation of Cyclic AMP-Generating Systems in Brain Membranes and Slices by the Diterpene Forskolin: Augmentation of Receptor-Mediated Responses

The diterpene forskolin markedly activates adenylate cyclase in membranes from various rat brain regions and elicits marked accumulations of radioactive cyclic AMP in adenine-labeled slices from cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, hypothalamus, thalamus, and medulla-pons. In cerebral cortical slices, forskolin has half-maximal effects at 20-30 microM on cyclic AMP levels, both alone and in the presence of the phosphodiesterase inhibitor ZK 62771. The presence of a very low dose of forskolin (1 microM) can augment the response of brain cyclic AMP-generating systems to norepinephrine, isoproterenol, histamine, serotonin, dopamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin does not augment responses to combinations of histamine-norepinephrine adenosine-norepinephrine, or histamine-adenosine. For norepinephrine and isoproterenol in rat cerebral cortical slices and for histamine in guinea pig cerebral cortical slices, the presence of 1 microM-forskolin augments the apparent efficacy of the amine, whereas for adenosine, prostaglandin E2, and vasoactive intestinal peptide, the major effect of 1 microM-forskolin is to increase the apparent potency of the stimulatory agent. In rat striatal slices, forskolin reveals a significant response of cyclic AMP systems to dopamine and augments the dopamine-elicited activation of adenylate cyclase in rat striatal membranes. The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor-mediated activation of the enzyme.     

Affinities of Histamine H1-Antagonists in Guinea Pig Brain: Similarity of Values Determined from [3H]Mepyramine Binding and from Inhibition of a Functional Response

Affinity constants for five antagonists at histamine H1-receptors in guinea pig brain have been determined from inhibition of the potentiation by histamine of the adenosine-induced accumulation of cyclic AMP in cerebral cortical slices. This action of histamine appeared to be mediated solely through H1-receptors. The affinity constants obtained were similar to those determined on peripheral H1-receptors and from inhibition of high-affinity [3H]mepyramine binding. This provides strong evidence that at least some of the [3H]mepyramine binding sites in guinea pig brain can be identified with functional H1-receptors.