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.     

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.     

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 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.     

ANTAGONISM BY DIBUTYRYL ADENOSINE CYCLIC 3',5'-MONOPHOSPHATE AND TESTOSTERONE OF CELL ROUNDING REACTIONS

In an attempt to understand further the mechanism of the morphological and functional "reverse transformation" of CHO-K1 cells induced by dibutyryl adenosine cyclic 3',5'-monophosphate (cAMP) and testosterone, the kinetics of variation in the susceptibility of cells to rounding after the addition or deletion of dibutyryl cAMP and testosterone have been investigated. Changes in susceptibility to cell rounding upon removal of divalent cations or pulse exposure to concanavalin A were complete within 0.5–1 h after addition or deletion of drug. In comparison, the gross conversion of CHO-K1 cells from epithelial- to fibroblast-like morphology after drug treatment or the converse change after drug removal required 8 or 4 h, respectively. The effects on cell rounding are not caused by an effect of dibutyryl cAMP upon cell growth rate. Inhibitor experiments indicate that the changes investigated do not require continued RNA or protein synthesis and are not prevented by agents which depolymerize microtubules.     

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 NUCLEOTIDES IN MURINE BRAIN: THE TEMPORAL RELATIONSHIP OF CHANGES INDUCED IN ADENOSINE 3',5'-MONOPHOSPHATE AND GUANOSINE 3',5'-MONOPHOSPHATE FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

–Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) levels increase about 5-fold in the cerebral cortex and 2-fold in the cerebellum following electroconvulsive shock (ECS). The peak levels of cyclic AMP occur at 45 s after ECS in the cerebral cortex, and at 15 s in the cerebellum. In the cerebral cortex, ECS produces twice the cyclic AMP accumulation as does decapitation in a comparable time period; however, the relative effect of a number of neurotropic agents on the cyclic AMP accumulation is essentially the same, whether stimulated by decapitation or by ECS. In the cerebellum, the levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP) also increase following ECS. The cyclic GMP levels are greatest at 60 s after ECS during the postictal depression. An association between elevated cerebellar cyclic GMP and depression seems unlikely, since CNS depressants either lowered or had no effect on cyclic GMP levels. From these results, cyclic nucleotide profiles following treatments such as ECS or decapitation may be useful in elucidating the molecular events involved in seizures, brain injury and ischemia.     

孕酮对小鼠全脑切片积聚3H-γ-氨基丁酸的影响

研究了孕酮对离体培养的小鼠全脑切片积聚＾３Ｈ－γ氨基丁酸的影响。结果表明,孕酮降低小鼠全脑切片对＾３Ｈ－氨基丁酸的积聚作用。该作用可被氨基酸Ａ受体激动剂蝇蕈醇加强,被氨基丁酸Ａ受体拮抗剂荷包牡丹碱和印防己毒素以及氨基丁酸Ｂ受体激动剂巴氯芬阻断,而且孕酮的效应以及各药物对其的影响均集中在０．０１－０．０５μｍｏｌ／Ｌ孕酮浓度中较为明显。     

β-Ar-ACETYL D-GALACTOSAMINIDASE IN BULK SEPARATED NEURONS AND NEUROPIL FROM RAT CEREBRAL CORTEX

Abstract— β- N -Acetyl D-galactosaminidase was studied in isolated neuronal and neuropil fractions from cerebral cortex and subcellular fractions derived from them. Although the enzyme activity evinced some latency properties, its subcellular distribution pattern was broader than that observed with other acid hydrolases. By contrast with nine other acid hydrolases, it was more active in neuropil than neuronal fractions (neuronal/neuropil activity ratio 0.63). This ratio was preserved in lysosomal subfractions derived from the isolated cell fractions. The data is taken as further evidence for the microheterogeneity of lysosomal particles from the brain.     

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.     

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.     

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.     

ADRENERGIC α- AND β-RECEPTORS EXPRESSED BY THE SAME CELL TYPE IN PRIMARY CULTURE OF PERINATAL MOUSE BRAIN

Abstract— The β-adrenergic agonist, isoproterenol and the α- and β-adrenergic agonist. NA. raise the intracellular concentration of cyclic AMP in cultures of dissociated perinatal mouse brain. This rise is prevented by a β- but not by an α-adrenergic antagonist. The maximal level of cyclic AMP reached in the presence of isoproterenol is markedly higher than that found after exposure to NA. However, if NA is used along with an α-adrenergic antagonist, cyclic AMP levels as high as those after isoproterenol are measured. Agonists with α-adrenergic activity including NA decrease the response to isoproterenol. The decrease is blocked by α-adrenergic antagonists. From this and additional evidence it is concluded: (1) The increase in the level of cyclic AMP caused by β-adrenergic agonists is due to β-receptor-mediated stimulation of adenylate cyclase; (2) the inhibition of this effect by α-adrenergic agonists is mediated by adrenergic α-receptors; (3) the α- and β-adrenergic receptors are likely to be located on the same cells, probably the most abundant putative glial precursor cells. The simultaneous stimulation of α- and β-adrenergic receptors on the same cell may be of significance in the regulation of the response to NA.     

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.     

THE EFFECT OF β,β''-IMINODIPROPIONITRILE AND ANAESTHESIA ON SOME ADENINE NUCLEOTIDES OF RAT BRAIN AND RETINA

—A significant increase in the retinal ATP content of anaesthetised rats was found 6 days after administration of β, β′-iminodipropionitrile (IDPN). With the development of retinal dystrophy variable ATP levels were observed from the 8th to the 12th day and low values were recorded on the 17th and 21st days. At 8 days after IDPN administration the ATP content of anaesthetised rat brain was significantly increased with slight decreases in ADP and AMP levels. The differences in the level of these adenine nucleotides in unanaesthetised and anaesthetised rat brain were not significant before or after IDPN administration. These results were related to previous experiments on the action of IDPN on the electroretinal response and the later development of a retinal microangiopathy. It was suggested that IDPN has a primary neurotoxic effect followed by the development of vascular morphological changes.     

EFFECTS OF DIAMINOPROPIONATE, DEOXYADENOSINE, AND THEOPHYLLINE ON STIMULATED FORMATION OF CYCLIC AMP AND GMP BY DEPOLARIZING AGENTS IN SLICES OF GUINEA-PIG CEREBRAL CORTEX

In incubated slices of guinea-pig cerebral cortex depolarizing agents such as veratridine and high potassium ions caused 50 to 80-fold increases of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) levels and these responses were inhibited about 50% by 2, 3-diaminopropionate and 2'-deoxyadenosine: the former is a specific antagonist for glutamate-elicited accumulation of cyclic AMP and the latter selectively for adenosine-elicited accumulation. Methylxanthines were powerful ‘inhibitors’toward the responses not only to depolarizing agents but also to glutamate and adenosine. These findings are consistent with the hypothesis that releases of both glutamate and adenosine are involved in the depolarization-elicited increases of cyclic AMP levels. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP) levels in the slices were also elevated by veratridine as well as by glutamate, but always to a lesser extent (8 ～ 12 times the control value) than cyclic AMP levels were. The responses for cyclic GMP both to veratridine and glutamate were ‘augmented’by methylxanthines and were not inhibited by 2, 3-diaminopropionate. Thus, glutamate appears to cause the increase of cyclic GMP levels through a different mechanism or site of action from that for cyclic AMP.