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: EFFECTS OF α- AND β-ADRENERGIC AGENTS, HISTAMINE, SEROTONIN AND ADENOSINE

—Norepinephrine and epinephrine, in combination with either adenosine or histamine, enhanced the accumulation of cyclic AMP in guinea pig cerebral cortical slices. Isoproterenol had only marginal effects under the same conditions. Studies with d- and l-norepinephrine and with the α- and β-adrenergic blocking agents, phenoxybenzamine, phentolamine, dihydroergokryptamine, propranolol and sotalol, indicated that the effect of catecholamines on cyclic AMP levels in this tissue was stereo-specific and was mediated primarily via interaction with a classical α-adrenergic receptor. Studies with the antihistaminics, diphenhydramine and pheniramine, and the antiserotonin agent, methysergide, indicated that guinea pig cerebral cortical slices contain receptors for histamine and serotonin, whose activation also stimulates an enhanced accumulation of cyclic AMP in the presence of adenosine.     

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.     

REGULATION OF CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE CONCENTRATION IN CHICK CEREBRAL HEMISPHERES DURING DEVELOPMENT

Abstract— Investigations have been carried out into developmental aspects of cyclic AMP metabolism and responsiveness to neurohormones in chick cerebral hemispheres. The in vivo cyclic AMP concentration, measured after freeze-blowing, was found to be highest in the embryonic brain, and changes in the cyclic nucleotide content produced by ischaemia increased with age. The magnitude of the in vivo increases in cyclic AMP produced by isoprenaline and by histamine decreased throughout the first postnatal month. The onset of isoprenaline- and histamine-induced cyclic AMP accumulation in brain slices occurred around 17 days embryonic age, reached a maximum at about 3 days post-hatch and fell to approx 50% of this response at 28 days of age. Adenosine stimulated cyclic AMP formation to a similar extent at all ages studied.
The activities of adenylate cyclase and cyclic AMP phosphodiesterase of hemisphere homogenates were found to reach maximum near the time of hatching. Since the overall pattern of responsiveness of the cerebral cyclic AMP system to neurohormones does not correlate with these variations in enzyme activities, it is suggested that changes occurring at the synaptic receptor level may explain the developmental variations observed.     

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.     

ACCUMULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE IN BRAIN SLICES: INTERACTION OF LOCAL ANAESTHETICS AND DEPOLARIZING AGENTS

Abstract— Membrane depolarizing agents such as veratridine, ouabain and high concentrations of potassium ions elicit a remarkable accumulation of cyclic AMP in brain slices incubated in vitro , and this accumulation, but not that elicited by biogenic amines, is prevented by a membrane stabilizer, cocaine. The effect of various local anaesthetics (compounds which are known to stabilize the membrane of peripheral sensory nerves) on the accumulation of cyclic AMP elicited by depolarizing agents in incubated slices of guinea pig brain has now been examined. At optimal concentrations the anaesthetics inhibited by more than 95 per cent the accumulation of cyclic AMP elicited with veratridine, ouabain, and high concentrations of potassium ions. The order of the inhibitory potency vs. veratridine was: dibucaine (ED₅₀= 9.5 ± 10⁻⁶ M) > tetracaine > cocaine (ED₅₀= 1·3 ± 10⁻⁴ M) > lidocaine > procaine (ED₅₀= 1.7 ± 10⁻³M). This order is consistent with the order of their local anaesthetic potency, but is not consonant with the order of the relative toxicity of these agents when used as spinal anaesthetics.     

CATECHOLAMINE INDUCED INCREASE OF CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE IN RAT BRAIN IN VIVO

Abstract— Four catecholamines injected into the cerebral ventricles increased the content of cyclic adenosine 3',5'-monophosphate (cAMP) in vivo in the whole brain of rats. The highest rise (2.6-fold) was measured 2 min after an injection of 100 μg epinephrine. Isoproterenol and norepinephrine were less active and dopamine hardly increased the cAMP level. These results are compatible with the view that physiological actions of catecholamines in the nervous system may be mediated by an increase of CAMP.     

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.     

ENZYMATIC REGULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE (CYCLIC AMP) IN THE FREEZING EPILEPTOGENIC LESION OF RAT BRAIN AND IN HOMOLOGOUS CONTRALATERAL CORTEX

Freezing epileptogenic lesions were made unilaterally in rat cerebral cortex. Such lesions were associated with an increase in adenyl cyclase activity and a decrease in the membrane-associated phosphodiesterase activity, with a concomitant increase in the level of cyclic AMP. Similar, though less striking, changes occurred in homologous contralateral cortex (‘mirror focus’). The effects of cyclic AMP on brain membrane systems are discussed, with the suggestion that this substance may play an important role in the genesis of focal seizures.     

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.     

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

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.     

BACLOFEN: EFFECTS ON AMINO ACID RELEASE AND METABOLISM IN SLICES OF GUINEA PIG CEREBRAL CORTEX

Slices of guinea-pig cerebral cortex were used to investigate the effects of the antispastic drug β-(p-chlorophenyl)-γ-aminobutyrate (Baclofen, Lioresal) on the release and metabolism of several amino acids. Electrical stimulation of slices evoked (1) a relatively large release, probably from nerve terminals, of ¹⁴C-labelled tissue glumate, aspartate and γ-aminobutyrate (GABA) synthesized via metabolism of D-[U-¹⁴C]glucose and (2) a relatively small release, probably not from nerve terminals, of ¹⁴C-labelled tissue alanine and threonine-serine-glutamine and of exogenous radiolabeled glutamate, aspartate, GABA and α-aminoisobutyrate that had been taken up from the medium. Baclofen (4μM) preferentially inhibited the release of ¹⁴C-labelled tissue glutamate and aspartate. It had no effect on the concentrations and specific radio-activities of most of the labelled tissue amino acids in the slices. However, it increased the turnover of ¹⁴C-labelled tissue glycine approx 4-fold and elevated the specific radio activity of tissue alanine by 40%. It was concluded that Baclofen affects transmission not by modulating the release of the inhibitory amino acid GABA, but by selectively suppressing the release of the excitatory amino acids glutamate and aspartate from nerve terminals. Provided that this action obtains in the spinal cord, it may at least partly underlie the antispastic action of Baclofen as glutamate and aspartate are presumed to be the transmitters released from terminals of non-nociceptive primary afferent fibers and excitatory interneurons, respectively. The Baclofen-induced increase in glycine turnover suggests an additional effect on inhibitory glycinergic interneurons in the spinal cord.     

PROTEIN PHOSPHORYLATION IN SYNAPTIC MEMBRANES REGULATED BY ADENOSINE 3'':5''-MONOPHOSPHATE: REGIONAL AND SUBCELLULAR DISTRIBUTION OF THE ENDOGENOUS SUBSTRATES

Abstract— The aim of these studies is the determination of the topographic distribution of the endogenous substrates in synaptic membrane preparations susceptible to phosphorylation in reactions modulated by adenosine 3':5'-monophosphate or Ca²⁺. To this end we have investigated the distribution of these phosphosubstrates in different cellular and synaptic subfractions obtained from rat cortex and corpus striatum, in other rat brain regions and in bovine retina, as well as membrane fractions from liver and testis.
The results obtained indicate that two cAMP-dependent protein substrates B (p80, i.e. apparent M _R= 80,000) and C (p75) are, as originally suggested by G reencard and collaborators confined to nerve tissue; they are located at least in part in the postsynaptic density, as is an additional protein D (p66). This localization appears to be shared by a cAMP-dependent, and a cAMP-independent entity, designated E (p54) and p48, respectively. In contrast, at least one prominent protein substrate requiring cAMP for its phosphorylation, component F (p50), appears to be associated predominantly with the postsynaptic membrane.
At least two of these cAMP-dependent (B and C), as well as two Ca²⁺ -dependent (p63 and p59), phosphoproteins appear to be associated with the presynaptic membrane.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

DIBUTYRYL CYCLIC ADENOSINE 3'' 5'' MONOPHOSPHATE, SUGAR TRANSPORT, AND REGULATORY CONTROL OF CELL DIVISION IN NORMAL AND TRANSFORMED CELLS

Swiss 3T3 cells exhibit contact-regulated cell growth and have a lower ability to transport 2-deoxyglucose than polyoma (Py)-transformed 3T3 cells. Py3T3 cells treated with dibutyryl cyclic adenosine 3'5' monophosphate (dBcAMP) and theophylline have reduced cell growth and transport 2-deoxyglucose at the same rate as normal 3T3 cells. Evidence that the cessation of cell growth and reduced transport abilities in Py3T3 cells does not represent a return to contact-regulated growth comes from the following observations. First, treating high density Py3T3 cells with dBcAMP allows more than two doublings of cell number, even though ability to transport 2-deoxyglucose is returned to levels equal to those of normal 3T3 cells. Second, dBcAMP prevents serum-stimulated increases in 2-deoxyglucose transport in Py3T3 but not in 3T3 cells.     

EFFECT OF β-ADRENERGIC DRUGS ON ADENOSINE 3'',5''-MONOPHOSPHATE IN RABBIT VAGUS NERVE

Abstract— Cyclic AMP was found to accumulate in rabbit vagus nerve after stimulation of specific β-adrenoceptors. The increase in cyclic AMP content by either isoproterenol or epinephrine was inhibited by the β-adrenoceptor antagonists sotalol and propranolol. α-Adrenoceptor agonists and antagonists, indirect sympathomimetics and theophylline had no effect on the accumulation of cyclic AMP in vagus nerve. The cyclic AMP increase caused by either β-adrenoceptor agents or adenosine was found to have no effect on resting potentials, action potentials or on post-tetanic hyperpolarization.     

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.     

COMPARISON OF LEVELS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON-NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA

Primary cultures containing ≥99% neurons, ≥99% non-neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12-day chick embryos. Levels of cyclic AMP in the non-neuronal cells (～14 pmol/mg protein) were approximately 3-fold higher than levels in the neurons (～4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non-neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non-neuronal cells. Thus, contact between the neurons and non-neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal-glial interactions, such as the specific neuronal stimulation of non-neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [³H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non-neuronal cells varied inversely with cell density. [³H]Thymidine incorporation was independent of cell density in both neuronal and non-neuronal cultures. Parallel density-dependent decreases in cyclic AMP concentration and [³H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non-neuronal cell proliferation and cyclic AMP levels in these cultures.     

THE MECHANISM OF THE EXCLUSION OF SODIUM IONS AND THE CONCENTRATION OF POTASSIUM IONS BY GUINEA-PIG CEREBRAL CORTEX SLICES

—Guinea pig cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline for periods of 1 s to 60 min, and their swelling and Na⁺ and K⁺ cone were measured. The swelling was at the rate of 8 per cent for the 1st min, and 0·8 per cent for the next 29 min; it fell significantly during the subsequent 30 min (P= 0·05). The Na⁺ and K⁺ concn in the tissue fluctuated during the 1st min of incubation, but the Na⁺ concn had risen to a mean of 108 mm after 1 min incubation and the K⁺ concn had fallen to a mean of 52 mm by 3 min. The concentrations of these cations did not change significantly after these times. Cerebral slices were also incubated for 30 min in isotonic media modified such that Na⁺, + K⁺, Na⁺+ choline⁺, or K⁺+ choline⁺ always added up to 150 mm . It was found that about half of the swelling (20-25 per cent) was independent of the Na⁺ or K⁺ concn and a further 20-25 per cent of the swelling varied with the cations only if Na⁺ and K⁺ were both present and was a function of the K⁺ concn in the medium (0·15 per cent m-mol). The Na⁺ concn in the tissue was a mean 8·4 mm after incubation in a Na⁺-free medium and 7·1 mm in K⁺ after incubation in a K⁺-free medium. Cerebral slices in the presence of Na⁺+ K⁺ excluded one molecule of Na⁺ for every four molecules in the incubating medium; they accumulated K⁺ from the medium until the concn in the medium exceeded 130 mm .