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.     

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.     

LOW CONCENTRATIONS OF LITHIUM INHIBIT THE SYNTHESIS OF CYCLIC AMP AND CYCLIC GMP IN THE RAT PINEAL GLAND

Abstract— Lithium chloride (2 m m ) significantly inhibited the increases in cyclic AMP and in cyclic GMP caused by norepinephrine or high concentrations of potassium in intact rat pineal glands. Adenylyl cyclase activity in homogenates and its stimulation by isoproterenol, a β-adrenergic agonist, were also inhibited. Lithium reduced the apparent V _max of isoproterenol-stimulated adenylyl cyclase activity without significantly affecting the apparent affinity for isoproterenol. There was no effect on the binding of the antagonist [³H]dihydroalprenolol to the β-adrenergic receptors, nor on the competition for binding sites by isoproterenol. Inhibition of adenylyl cyclase activity by lithium was inversely related to the magnesium concentration in the reaction mixture. There was no differential effect of lithium on adenylyl cyclase activity from supersensitive vs subsensitive glands. Lithium may inhibit cyclic nucleotide synthesis by interfering with the role of divalent cations.     

COMPARTMENTATION OF AMINO ACID METABOLISM IN THE RAT POSTERIOR PITUITARY

—Isolated rat posterior pituitary glands were incubated with [¹⁴C]glucose or [¹⁴C]acetate and the incorporation of radioactivity into several amino acids was followed. The results indicated that radioactivity was incorporated from [¹⁴C]glucose into a large pool of glutamate which appeared to be responsible for a large proportion of GABA synthesis in the gland. The specific activity of glutamine was always less than that of glutamate when [¹⁴C]glucose was the precursor employed, whereas [¹⁴C]acetate labelled a glutamate pool which had approximately the same specific activity as that of glutamine. The results are discussed with reference to the compartmentation of amino acid metabolism in the nervous system.     

损毁和刺激垂体对大鼠痛阈的影响

采用局限性损毁和刺激垂体的方法,以行为测痛为指标,观察大鼠垂体在痛觉调节中的作用以及地塞米松（Ｄｅｘ）对其影响。实验结果显示,损毁垂体中间叶（ＩＬ）及邻近的前叶（ＡＬ）,大鼠痛阈明显低于手术前的痛阈（Ｐ＜０．０１）。电刺激垂体的上述同样部位,大鼠痛阈明显高于手术基础值及自身假刺激值（Ｐ＜０．００１）。经Ｄｅｘ处理的动物,电刺激垂体不再引起痛阈升高。结果表明,大鼠垂体ＩＬ及靠近ＡＬ与痛调节有关,这种     

ROLE OF ADENOSINE 3'', 5''-MONOPHOSPHATE IN THE REGULATION OF CIRCADIAN OSCILLATION OF SEROTONIN N-ACETYLTRANSFERASE ACTIVITY IN CULTURED CHICKEN PINEAL GLAND

—When pineal glands of 10–12-day-old chicks were organ-cultured in darkness, serotonin N-acetyltransferase activity was low during the daytime, increased at midnight and then decreased to the daytime level the next morning. The pattern of increase and decrease of enzyme activity in cultured pineal glands was comparable to the circadian rhythm of N-acetyltransferase activity in vivo. When pineal glands were kept at a low temperature for 5 h prior to culture, the phase of autonomous rhythm of enzyme activity was delayed. When chicken pineal glands were cultured during the daytime for 6 h, derivatives of adenosine 3′, 5′-monophosphate (cyclic AMP), cholera toxin, a high concentration of KCl and phosphodiesterase inhibitors increased N-acetyltransferase activity 3–7-fold, indicating an involvement of cyclic AMP in the regulation of N-acetyltransferase activity in chicken pineal gland as has been shown in rat pineal gland. When pineal glands were cultured at night in darkness, cholera toxin or a high KCl did not enhance the night-time increase of the enzyme activity. Derivatives of cyclic AMP or phosphodiesterase inhibitors enhanced the autonomous night-time increase of N-acetyltransferase activity in an additive or more than additive manner in cultured pineal glands. These observations suggest that adenylate cyclase of pinealocytes is inactive during daytime, but is activated at night in darkness, which is transduced to the synthesis of N-acetyltransferase molecules. Catecholamines suppressed the basal level and the nocturnal increase of N-acetyltransferase activity via α-adrenergic receptor. The nocturnal increase of enzyme activity was prevented by cycloheximide or actinomycin D. Cocaine, which stabilizes cell membrane potential or light exposure, blocked the nighttime increase of N-acetyltransferase activity in cultured chicken pineal glands.     

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.     

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.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

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.     

大鼠垂体细胞ACTH分泌的调节控制

本文建立了大鼠垂体细胞灌流系统并利用这系统和放射免疫测定法检测了一些物质对垂体细胞促进或抑制ACTH的分泌作用。下丘脑抽提液能刺激垂体细胞分泌ACTH,并有明确的剂量反应关系;AVP,cAMP,Ca~(2+),K~+,去甲肾上腺素、灭吐灵和氟哌啶醇等对垂体细胞ACTH分泌也有兴奋作用。地塞米松和多巴胺能抑制垂体细胞ACTH的基础分泌并对抗兴奋性物质的作用;赛庚啶能选择性地拮抗某些兴奋性物质的作用,γ-氨基丁酸无明显抑制作用。     

ELECTROPHORETIC ANALYSES OF PROTEINS TRANSPORTED TO THE RAT POSTERIOR PITUITARY

Abstract— [³⁵S]cysteine, [³H]methionine, or [³H]fucose were injected into the supraoptic nuclei (SON) of rats, and the labelled proteins that were transported to and accumulated in the posterior pituitary 24h post-injection were analyzed electrophoretically. The transported, labelled proteins which were soluble in 0.1 m -HCl were primarily of low molecular weight (about 12,000 on SDS gels). However, the selectivity of labelling of these proteins by the three different labelled precursors could be revealed by isoelectric focusing. The 0.1 m -HCl insoluble labelled proteins, presumably reflecting membrane proteins transported from the SON to the pituitary, were more diverse and generally of higher molecular weight (> 43,000 on SDS gels).     

POSTSYNAPTIC ADRENERGIC-CYCLIC AMP CONTROL OF THE SEROTONIN CONTENT OF CULTURED RAT PINEAL GLANDS

Abstract— This investigation was designed to determine whether the amount of serotonin (5-HT) in cultured pineal glands can be altered by norepinephrine (NE). Treatment with l -NE (10^?5-10^?7m ) for 4-6 h caused a gradual decrease in the concentration of 5-HT to a value that was less than 30% of that in the untreated control gland. This effect was observed using chronically denervated pineal glands. d -Norepinephrine (10^?6-10^?7m ) and dopamine (10^?4m ) were ineffective in lowering 5-HT. The effect of l -NE was completely blocked by a β-adrenergic receptor blocker, propranolol and was only slightly decreased by α-adrenergic receptor blockers. These observations indicate that l -NE acts post-synaptically via a highly specific β-adrenergic mechanism. The effect of l -norepinephrine was mimicked by theophylline and N⁶, 2′0-dibutyryl adenosine 3′,5′-monophosphate, an indication that adenosine 3′,5′-monophosphate is involved in the effect of l -NE on 5-HT. Treatment with cycloheximide, which by itself caused a decrease in pineal 5-HT, also blocked any further decrease caused by treatment with l -NE, an indication that protein synthesis is necessary for maintenance of baseline levels of serotonin and for the effect of l -NE to be observed. The total amount of l -[³H]NE and degradation products of L-[³H]NE in the gland after 6 h of treatment with l -[³H]NE was less than 3 pmol. This amount of l -NE and degradation products of l -NE could not account for the decrease of 100-200 pmol of 5-HT on the basis of a mole for mole replacement of 5-HT by l -NE. These findings are consistent with the hypothesis that non-neuronal pineal 5-HT is physiologically regulated by the release of l -NE from the sympathetic nerve network.     

SUBCELLULAR LOCALIZATION OF TRYPTOPHAN-5-MONO-OXYGENASE IN BOVINE PINEAL GLANDS AND RAPHE NUCLEI

Abstract— Tryptophan-5-mono-oxygenase from both bovine raphe nuclei and pineal glands was activated by preincubation with dithiothreitol and ferrous ion at pH 8.5. The optimum pH for the enzyme activity lies between pH 6.4 and 7.3. Preincubation increased the activity of the enzyme from raphe nuclei by about 20 times in both the homogenate and 105,000 g precipitate prepared from it. Activity in the 105,000 g supernatant fraction was about trebled. Corresponding increases in pineal gland enzyme activity were noted: 100 times in homogenate and 105,000 g precipitate and 15 times in 105,000 g supernatant fluid. Total recoveries of activated enzyme from the homogenate prepared in hypo-osmotic medium, in the 105,000 g supernatant and precipitate, were 87.1% and 79.0% for raphe nuclei and pineal glands respectively. Of this, 89.5-91.3% in the case of the raphe nuclei and 76.0-82.0% in the case of the pineal glands, was found in the precipitate. In contrast, 85-90% of the lactate dehydrogenase activity was found in the supernatant fraction. The results of subcellular fractionation revealed that the raphe nuclear enzyme was located in both 'mitochondrial' and 'microsomal' fraction while the pineal gland enzyme was effectively restricted to the 'mitochondrial' fraction. The structural characteristics of the fraction were confirmed by electron microscopy.     

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.     

ISOLATION AND BIOCHEMICAL STUDY OF SECRETORY GRANULES FROM RAT PITUITARY GLANDS

Secretory granules from anterior pituitary glands of young adult male castrate rats were isolated by differential centrifugation, microfiltration, and discontinuous density gradient centrifugation. The granules were obtained as pellets, sectioned, and studied with the electron microscope. A major part of the gonadotropin and a substantial amount of the TSH were associated with the isolated granules. Negligible amounts of growth hormone and prolactin were present as contaminants. Succinic dehydrogenase, glucose-6-phosphatase, acid protease, and acid and alkaline phosphatases were not found in the granules. Alkaline protease was the only enzyme found to be associated with the granules, and it is suggested, in the light of these results, that the alkaline protease may be involved in the release of the hormones.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.