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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

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.     

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.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

A RADIOISOTOPIC METHOD FOR MEASURING THE FORMATION OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN INCUBATED SLICES OF BRAIN

Abstract— A simple and sensitive method for measuring the effect of neurohormones and other chemical agents on the formation of adenosine 3',5'-cyclic monophosphate (cyclic 3',5'-AMP) in incubated slices of brain was developed. The principle of the method depends on pulse-labelling of adenosine-5'-triphosphate in slices of brain with [8-¹⁴C]adenine, followed by incubation in a medium containing the test substance, separation by thin-layer chromatography of the cyclic nucleotide formed in the slices, and radioassay. The purity of the cyclic nucleotide was confirmed by chromatography in a variety of systems and by hydrolysis with a specific, bovine-heart phosphodiesterase. The method was used to study the effect of histamine, norepinephrine, and adenosine on the accumulation of adenosine 3',5'-cyclic monophosphate in incubated slices of brain.     

EFFECT OF DEXAMETHASONE ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE IN CHROMAFFIN TISSUE OF THE NEONATAL RAT

Abstract— Treatment of neonatal rats with dexamethasone resulted in the appearance of phenylethanolamine- N -methyltransferase (PNMT) and numerous small, intensely fluorescent (SIF) cells in abdominal paraganglia and in sympathetic paravertebral ganglia. These cells may be derived from a primitive stem cell precursor, but because of their unusual anatomical features, origin from ganglion cells cannot be altogether ruled out. Associated with the proliferation of the cells was a marked increase in the PNMT activity of the tissues. The PNMT response to the glucocorticoid was limited to the first few days of life, as was the SIF cell response. After discontinuance of dexamethasone, the enzyme activity fell very rapidly, while the number of cells declined at a slower rate.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: EFFECT OF HYPOTHERMIA ON ADENOSINE 3'',5'' MONOPHOSPHATE, GLYCOGEN PHOSPHORYLASE, GLYCOGEN SYNTHASE AND METABOLITES FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

Abstract —The accumulation of adenosine-3',5'-cyclic monophosphate (cyclic AMP) has been investigated in murine brain following electroconvulsive shock and decapitation. Animals were made hypothermic (20°C) to minimize the freezing time of the brain and to delay metabolic events. Cyclic AMP concentrations were decreased in the cerebral cortex of hypothermic rats and mice. Furthermore, the changes in cyclic AMP elicited by electroconvulsive shock and decapitation were delayed. In hypothermic animals, the metabolic rate as determined by high energy phosphate use was decreased to 65% of control values. The interconversions of the active and inactive forms of glycogen phosphorylase and glycogen synthase were sufficiently retarded in hypothermic animals to correlate with changes in cyclic AMP concentrations. The conversion of phosphorylase b to a and synthase a to b occurred when cyclic AMP concentrations had increased from 2 to 5 μmol/kg, following either electroconvulsive shock or decapitation. The results indicate that cyclic AMP plays a role in regulation of glycogen metabolism in cerebral cortex.