Cerebral Cortical Slices Research Articles

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

The effect of Ca 2+ 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 Ca 2+ 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 E 1 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 Ca 2+, norephinephrine and adenosine.

Cyclic AMP Accumulation in Cerebral Cortical Slices: Effect of Carbamazepine, Phenobarbital, and Phenytoin

Recent investigations have suggested that abnormal increases in brain cyclic 3',5'-adenosine monophosphate (cAMP) may play a role in epileptogenesis. Therefore, the effect of three commonly used antiepileptic drugs on cAMP accumulation in rat cortex slices was investigated. Ouabain, a depolarizing agent which produces seizures when applied to rat cortex, produced a five- to sevenfold increase in cAMP accumulation, and both carbamazepine and and phenytoin inhibited this increase. Ouabain stimulation may be mediated by the release of endogenous adenosine, and carbamazepine antagonized adenosine stimulation of cAMP accumulation whereas phenytoin did not. Carbamazepine had no effect on adenosine efflux. The augmentation of cAMP accumulation by norepinephrine was inhibited by carbamazepine and phenobarbital but slightly increased by phenytoin. If increases in brain cAMP are involved in epileptogenesis, the antagonism of cAMP accumulation by antiepileptic drugs may play a role in their anticonvulsant action.

Effect of bufalin on acetylcholine levels in rat brain: relation to its CNS-stimulating action.

1. Total acetylcholine (ACh) levels in the rat whole brain homogenate decreased during the maximal tonic extension induced by bufalin. 2. "Free" ACh levels in the rat whole brain homogenate increased during the maximal tonic extension induced by bufalin, but not by pentylenetetrazol. 3. ACh release from rat cerebral cortical slices increased in a medium containing bufalin. 4. After intracerebral Hemicholinium-3 or scopolamine, 50% convulsive dose of bufalin in mice was increased, but was decreased after eserine. 5. A relationship between the convulsant effect of bufalin and its ACh-releasing effect was discussed.

Effect of external ACh and of atropine on 14C-ACh synthesis and release in rat cortical slices

1. The effects of extracellular ACh and of atropine on the changes in 14C-ACh release and synthesis were examined in rat cerebral cortical slices using U-14C-glucose as the precursor of the ester's acetyl-moiety. The release and the synthesis of the labelled ester were measured in the presence of eserine during a 10 min period of depolarization (incubation) produced by 31 mM KCl, following a 60 min preincubation with 5.6 mM KCl. 2. The rates of 14C-ACh synthesis and release were low when the ACh released in the course of the preincubation was still present during the 10 min incubation. They were in contrast much higher in two other experimental situations tested in which no extratissular ACh was present at the onset of incubation, i.e., when the preincubation was conducted in the absence of eserine or when the preincubation medium was discarded by filtration. The effect of the extracellular ACh on the 14C-ACh synthesis and release processes is mediated through a muscarinic receptor. Indeed, this inhibitory effect of external ACh could be reversed when atropine (10 μM) was present during the incubation period. 3. The respective contributions, during the 10 min incubation, of the “newly synthesized” 14C-ACh-i.e. the labelled molecules formed during the incubation period-, and of the “stored” 14C-ACh-i.e. those molecules remaining in the tissues at the end of the preincubation-, to the release process evoked by 31 mM KCl were studied in two separate experiments, in which 14C-glucose was added only during the final incubation or only during the preincubation, respectively. It was found that only 40% of the KCl (31 mM) evoked release originated from the “newly synthesized” fraction, the rest deriving from the “stored” 14C-ACh. This proportion was not affected when atropine was present during the incubation period. It is concluded from these results that the “newly synthesized” ACh is not preferentially released under the experimental condition used, and that the inhibitory mechanism, triggered by the external ACh, on the rates of 14C-ACh synthesis and release, affects both the “newly synthesized” and the “stored” 14C-ACh. 4. When similar experiments were carried out with 8 mM KCl, atropine (10 μM) stimulated solely the release of the 14C-ACh synthesized during the final 60 min incubation proceeding in the presence of the depolarizing agent. Indeed, atropine did not potentiate the 8 mM KCl-evoked release of “stored” 14C-ACh. Atropine (10 μM) significantly increased the release of 14C-ACh formed from 14C-glucose during a 60 min incubation made with a normal concentration of KCl (5.6 mM). 5. These various results support the concept of an important role of presynaptic muscarinic receptors in the control of ACh synthesis and release in central cholinergic neurons.

Strain differences in responsiveness of norepinephrine-sensitive adenosine 3′,5′-monophosphate-generating systems in rat brain slices after intraventricular administration of 6-hydroxydopamine

The development of hyperresponsiveness in cyclic AMP-generating has been investigated in brain slices of Sprague-Dawley and F-344 rats following intraventicular administration of 6-hydroxydopamine. Hyperresponsiveness to adrenergic agonists in cerebral cortical slices of Sprague-dawley rats pertained during the period 5–25 days after treatment with 6-hydroxydopamine. In contrast, hyperresponsiveness did not develop in cerebral cortical slices of F-344 rats. Reductions in norepinephrine levels of the cerebral cortex and hypothalamus following 6-hydroxydopamine treatment were comparable in the two rat strains. A hyperresponsiveness to norepinephrine and isoproterenol failed to develop in mesencephalic slices of either strain 12–14 days after treatment with 6-hydroxydopamine. The accumulation of cyclic AMP elicited by Norepinephrine in cortical slices of F-344 rat is normally about 50% greater than the accumulation elicited in slices from Sprague-Dawley rats. However, after 6-hydroxydopamine treatment, there was no significant difference in the accumulations of cyclic AMP elicited in cortical slices from the two rat strains. These data indicate there may be a limit to the responsiveness of catecholamine-sensitive cyclic AMP generating systems which can develop following alterations in synaptic input.

Behavioral activity and accumulation of cyclic AMP in brain slices of strains of mice

Accumulations of cyclic AMP elicited by norepinephrine, adenosine and a norepinephrine-theophylline combination were measured in cerebral cortical slices from several inbred mouse strains. There were no apparent correlations between the responses of cyclic AMP-generating systems and active avoidance learning for seven strains. Adenosine-elicited accumulation of cyclic AMP and sponteneous behavioral activity did show an inverse correlation in four inbred mouse strains. In individual mice of a randomly bred strain, ability to learn an avoidance task appeared inversely correlated with responsiveness of cortical cyclic AMP-generating systems to norepinephrine, but showed no significant correlation with responsiveness to adenosine.

Interaction of clonidine with pre- and post-synaptic adrenergic receptors of rat brain: Effects on cyclic AMP-generating systems

The locus and mechanism of interaction of clonidine with catecholamine-elicited accumulations of cyclic AMP has been investigated in brain slices from control and 6-hydroxydopamine-treated rats of the F-344 and Sprague—Dawley strains. The inhibitory effects of clonidine on the norepinephrine-stimulated accumulation of cyclic AMP and the potentiative effects of clonidine on the isoproterenol-stimulated accumulation of cyclic AMP are present to the same extent in cerebral cortical slices from control and 6-hydroxydopamine-treated Sprague—Dawley rats. Clonidine, at concentrations between 0.001 and 100 μM has no intrinsic stimulatory activity on cortical cyclic AMP-generating systems from either control or 6-hydroxydopamine-treated rats. In F-344 rats phenoxybenzamine (100 μM) elicits a significant accumulation of cyclic AMP in cerebral cortical slices which can be abolished by pretreatment of rats with 6-hydroxydopamine, or by incubation of tissue slices with either clonidine or sotalol. The hyperresponsiveness of catecholamines usually observed following central administration of 6-hydroxydopamine failed to develop in the F-344 rat. Concentrations of phenoxybenzamine which have no significant stimulatory effects on cyclic AMP accumulation are capable of abolishing the potentiative effects of clonidine on the isoproterenol-stimulated formation of cyclic AMP. The β-antagonist, sotalol, is a less effective antagonist of isoproterenol-stimulated accumulation of cyclic AMP in the presence of clonidine. Clonidine has no significant effect on the accumulation of cyclic AMP elicited by submaximal concentrations of isoproterenol in cerebellar slices. In toto, the data are consistent with the hypothesis that both the inhibitory effects on norepinephrine-stimulated accumulation of cyclic AMP and the stimulatory effects of clonidine on isoproterenol-elicited accumulation of cyclic AMP are exerted at postsynaptic α-adrenoceptors. No evidence was found for a presynaptic generation of cyclic AMP, although clonidine does reverse the stimulatory effects of phenoxybenzamine on cyclic AMP accumulation, presumably by interaction with a presynaptic site controlling norepinephrine release.

A MODULATING ROLE OF PITUITARY‐ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3′,5′‐MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3′,5′‐monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3′.5′‐nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.

Release of14C-adenine derivatives from cerebral cortical slices: Effect of phenytoin and phenobarbital

Both ouabain, 0.1 mM, and veratridine, 0.05 mM, increased the release of(14)C-labeled compounds from rat cortical slices prelabeled with(14)C-adenine and incubated in vitro. The increment in radioactivity released by both depolarizing agents was almost entirely a result of increases in adenosine, inosine, and hypoxanthine. However, the distribution of these three compounds in the ouabain-induced efflux (adenosine, 12%; inosine, 51%; hypoxanthine, 36%) contrasted with that evoked by veratridine (adenosine, 42%; inosine, 15%; hypoxanthine, 38%). Phenytoin significantly reduced the efflux of(14)C-labeled compounds produced by both ouabain and veratridine, but phenobarbital had no effect. The intracortical injection of adenosine, inosine, and hypoxanthine has been shown to induce epileptiform discharges in rats, and it is suggested that the inhibitory effect of phenytoin on the release of adenine derivatives may play a role in its antiepileptic action.

Antagonism of α- and β-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the β-antagonist (−)alprenolol

(−)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10 −5M. Other β-adrenergic antagonists such as (−)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (−)alprenolol does not exhibit absolute specificity for β-receptors.

The uptake ofΔ 9-tetrahydrocannabinol in choroid plexus and brain cortex in vitro and in vivo

[3H]delta9 Tetrahydrocannabinol (delta9-THC) was actively transported by the choroid plexus and cerebral cortical slices of the rabbit when incubated as a BSA-microsuspension in artificial rabbit CSF. The transport system for delta9-THC in choroid plexus had a V max of 174 nmoles/mg tissue/h, approximately 9-fold greater than that observed for cortical slices. In vivo experiments demonstrated a preferential distribution of delta9-THC in choroid plexus at 1 h after intravenous injection. These results indicate that delta9-THC is actively accumulated by choroidal epithelium and may also be transported across the epithelial stroma into the capillary circulation. This suggests that the choroid plexus participates in the regulation of delta9-THC concentration in CSF and indirectly in brain by means of the "sink" function of the CSF.

Adenosine-dependent formation of cyclic AMP in brain slices

Methylxanthines, adenosine analogs, phosphodiesterase inhibitors and adenosine deaminase have been used to assess the role of adenosine in stimulation of radioactive cyclic AMP formation by ATP, cyclic AMP, glutamate, veratridine, ouabain, histamine and histamine-norepinephrine combination in [ 14C]adenine-labeled guinea pig cerebral cortical slices. Theophylline and isobutylmethylxanthine effectively antagonized the response to adenosine, ATP, cyclic AMP, an adenosine-histamine combination, glutamate and veratridine, had lesser effects on the response to ouabain and the histamine-norepinephrine combination and had no significant antagonistic effect on the histamine response. 2′-Deoxyadenosine had a similar profile of antagonistic effects with the exception of the glutamate response where it had no antagonistic action. Adenine xylofuranoside was an effective antagonist of responses to all agents, a result suggestive of a non-specific blockade of adenylate cyclases. Adenosine deaminase was an effective antagonist of the response to adenosine, ATP, cyclic AMP, an adenosine-histamine combination, veratridine and glutamate, but also partially antagonized the response to histamine and the histamine-norepinephrine combination. The phosphodiesterase inhibitors, papaverine, RO 20–1724 and diazepam, potentiated the response to amines. The latter two compounds also potentiated the response to adenosine and glutamate. The results suggest a role for adenosine in responses to most agents in brain slices and indicate that 2′-deoxyadenosine may be a more specific adenosine antagonist than either methylxanthines or adenosine deaminase.

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1, N 6-ethano[ 14C]adenosine nor 1, N 6-ethanol[ 14C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8- 3H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N 6-Benzyl[ 14C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N 6-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8- 14C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.

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 Withdrawal from Chronic Ethanol Ingestion on the cAMP Response of Cerebral Cortical Slices Using the Agonists Histamine, Serotonin, and Other Neurotransmitters

The effect of ethanol withdrawal on the cAMP response of cerebral cortical brain slices was studied. The cAMP response was evoked in vitro by various neurotransmitters including norepinephrine (NE), histamine, serotonin, dopamine, acetylcholine, and gamma-aminobutyric acid (GABA). The cAMP response to NE and histamine was enhanced by ethanol withdrawal. Serotonin evoked a cAMP response in the brain slices from ethanol-withdrawal rats but not in pair-fed controls. The histamine and serotonin evoked responses were blocked by chlortripolon and methysergide, respectively. The responses to histamine and serotonin were also blocked by alpha- and beta-adrenergic antagonists, possibly because of the nonspecific membrane stablizing effect of these antagonists. GABA inhibited the NE stimulated cAMP response possibly through the hyperpolarizing action of GABA. The results support the hypothesis that ethanol withdrawal induces a nonspecific postjunctional supersensitivity. It is postulated that the supersensitivity involves a partial depolarization of the receptor membrane. Alternative hypotheses are reviewed.

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

Abstract—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.

FUNCTIONAL COMPARTMENTS OF ADENINE NUCLEOTIDES SERVING AS PRECURSORS OF ADENOSINE 3′,5′‐MONOPHOSPHATE IN MOUSE CEREBRAL CORTEX

Abstract—Cyclic AMP accumulates in cerebral cortical slices from the C57B1/6J mouse incubated with the following stimulatory agents: norepinephrine, adenosine, veratridine and adenosine‐biogenic amine combinations. The results with slices labelled with radioactive adenine or adenosine provide evidence for the existence of distinct functional compartments of adenine nuclcotides which serve as precursors of cyclic AMP on stimulation with specific agents. Thus, in slices labelled with [14C]adenine or [3H]adenosine the ratio of [14C] to [3H]cyclic AMP was dependent on the stimulatory agent; with veratridinc the ratio was 1.4 while with adenosine the ratio was 3.0. In addition, a greater than 2‐fold difference in the ratio of endogenous/radioactive cyclic AMP was observed in adenine or adenosine‐labelled slices after incubation with veratridine, norepinephrine, adenosine or adenosine‐amine combinations; the lowest ratios after stimulation with veratridine and the highest after adenosine or adenosine‐amine combinations. The high ratio observed with adenosine was in part due to a quite marked incorporation of the stimulant, adenosine, into the accumulating cyclic AMP. Such distinct functional compartments of cyclic AMP precursors may represent different cell types and/or morphological entities within one cell type.

Stimulation of adenosine 3?,5?-monophosphate formation in guinea-pig cerebral cortical slices in a calcium free medium

In guinea-pig cerebral cortical slices cyclic AMP concentrations increase during incubation with histamine+noradrenaline. After 10 min of incubation the levels of cyclic AMP start to decline. When calcium ions are omitted from the incubation medium, cyclic AMP levels do increase to a greater extent under the same conditions and do not drop during 30 min incubation. In the presence of calcium ions cyclic AMP synthesis can not be elicited by noradrenaline alone. In calcium-free Krebs-Ringer solution a pronounced effect of noradrenaline on cyclic AMP levels is observed. This effect of noradrenaline is shown to be mediated by a classical alpha-type receptor. 5-Hydroxytryptamine, prostaglandin E1 and dopamine do not significnatly enhance cyclic AMP formation in guinea-pig brain slices in either the presence in, or the absence of calcium ions from the incubation medium. Under depolarizing conditions of incubation the stimulatory effect of ouabain or 125 mM K+ is blocked in a calcium-free medium, while with the depolarizing agent veratridine no significant reduction of cyclic AMP formed during incubation in a calcium-free medium is obtained.

The release of labelled acetylcholine and choline from cerebral cortical slices stimulated electrically.

1 In order to establish the origin of the increased efflux of radioactivity caused by electrical stimulation of cerebral cortical slices which had been incubated with [(3)H]-choline, labelled choline and acetylcholine (ACh) collected by superfusion were separated by gold precipitation.2 In the presence of physostigmine electrical stimulation (1 Hz, 10 min) increased the release of only [(3)H]-ACh which was greatly enhanced by the addition of atropine.3 Continuous stimulation in the presence of physostigmine resulted in an evoked release of [(3)H]-ACh which declined asymptotically. This evoked release appeared to follow first-order kinetics with a rate constant which remained stable over the course of prolonged stimulation.4 The rate constant for the evoked release of [(3)H]-ACh with 1 Hz stimulation was three times greater in the presence of physostigmine and atropine than in the presence of physostigmine alone, while the size of the store from which [(3)H]-ACh was released was nearly identical under these two conditions.5 In the absence of physostigmine and atropine, stimulation caused the appearance of only [(3)H]-choline in the samples.6 Reduction of [(3)H]-ACh stores before the application of physostigmine resulted in a reduced evoked release of total radioactivity, both in the absence or presence of physostigmine and atropine, and decreased the evoked release of [(3)H]-ACh without affecting the release of [(3)H]-choline.7 Results suggest that electrical stimulation of cortical slices which had been incubated with [(3)H]-choline causes the release of only [(3)H]-ACh, both in the presence or absence of an anticholinesterase. The evoked increase in the efflux of total radioactivity is therefore a good measure of the release of [(3)H]-ACh.

Stimulated Formation of Cyclic Adenosine 3′:5′-Monophosphate by Aspartate and Glutamate in Cerebral Cortical Slices of Guinea Pig

Abstract The content of cyclic adenosine 3':5'-monophosphate (cyclic AMP) in guinea pig cerebral cortical slices increases approximately 40-fold during a 10-min incubation and 80-fold during a 20-min incubation in a medium containing 10mm l-glutamate. A similar increase was observed in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides, labeled by a prior incubation with radioactive adenine. The stimulatory effect of l-glutamate on formation of radioactive cyclic AMP was found with a concentration as low as 0.05 mm, and concentrations of l-glutamate required for the half-maximal and maximal effects were about 1.5 and 10 mm, respectively. At 10 mm concentration, l-aspartate was as good as, and d-aspartate, d-glutamate, dl-α-aminoadipate, and l-alanine were weaker than, l-glutamate as a stimulant for the cyclic AMP formation. All other amino acids and metabolites of glutamic acid tested were virtually ineffective. Mutual potentiation of effects was observed when aspartate was added together with either adenosine or histamine, but not with glutamate, veratridine, or ouabain. The effect of the two amino acids were completely blocked by theophylline, but not by omission of Ca2+ or by addition of cocain, the conditions known to inhibit the depolarization-elicited formation of cyclic AMP. The formation of radioactive cyclic AMP stimulated by l-glutamate was allowed to return to a half of the stimulated levels within 20 min after removal of the glutamate by washing, and then was restimulated either by adenosine, l-aspartate, or l-glutamate. These properties of the amino acids suggest that the amino acids may be a new class of stimulants which is different from biogenic amines, adenosine, and depolarizing agents as stimulants for cyclic AMP formation in incubated brain slices.