Comparison of the effects of biogenic amines on cyclic GMP and cycle AMP levels in mouse cerebellum in vitro.

Norepinephrine (NE) elevates levels of both 3',5'-guanosine monophosphate (cyclic GMP) and 3',5'-adenosine monophosphate (cyclic AMP) in incubated slices of mouse cerebellum. As little as 1 muM NE is capable of increasing the level of either cyclic nucleotide. Maximal elevations of cyclic AMP and cyclic GMP levels produced by NE are 15- to 40-fold and 2- to 4-fold, respectively. Dopamine, serotonin and histamine, other biogenic amines considered to be neurotransmitters in CNS, have no effect on mouse cerebellum cyclic nucleotide levels except at relatively high (1 mM) concentrations. NE-induced accumulation of cyclic GMP, but not cyclic AMP, is blocked by omission of Ca2+ from the incubation media. Theophylline does not alter the effect of this catecholamine on either cyclic nucleotide. In tissue slices incubated in buffered sucrose or in choline-Krebs buffer, NE is still capable of increasing both cyclic GMP and cyclic AMP levels; however, these elevations are less than those observed in brain slices incubated in Krebs-Ringer buffer. NE, in combination with glutamate, produces supra-additive elevations of both cyclic GMP and cyclic AMP levels. However, NE in combination with adenosine or high levels of K+ only has a synergistic effect on cyclic AMP and not on cyclic AMP and not on cyclic GMP combination. The elevation of cyclic AMP levels produced by NE appears to be mediated via both alpha- and beta-adrenergic receptor sites. In contrast, the receptor site(s) mediating cyclic AMP accumulation do not appear to be either typical alpha- or beta-adrenergic receptors.     

Relationships between seizure activity and cyclic nucleotide levels in brain

The effects of pentylenetetrazol on behavior, EEG activity and regional CNS levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) in mice and guinea pigs were studied. Pentylenetetrazol increased cGMP levels in all regions of brain examined (cerebral cortex, hippocampus, striatum and cerebellum) and increased cAMP levels in all regions except striatum. cGMP levels were increased by both sub-convulsant and convulsant doses of pentylenetetrazol. In contrast, cAMP levels were elevated only by concentrations of pentylenetetrazol that produced clinically evident seizures or epileptiform EEG activity. These data indicate that increases in CNS cGMP levels produced by epileptogenic stimuli can occur independently of seizure discharges, whereas accumulation of cAMP requires and is secondary to seizure activity. In conjunction with results of other studies, these data support the hypothesis that cGMP may have a role in seizure genesis and/or propagation, whereas cAMP may be involved in processes that attenuate or terminate seizures.     

Inositol trisphosphate, cyclic AMP, and cyclic GMP in rat brain regions after lithium and seizures.

The mechanism of action of lithium, the primary treatment for bipolar affective disorder, is unknown but may involve inhibition of second messenger production in the brain. Therefore, the concentrations of three second messengers, inositol 1,4,5 trisphosphate (Ins 1,4,5P3), cyclic adenosine monophosphate (AMP), and cyclic guanosine monophosphate (GMP), were measured in rat cerebral cortex and hippocampus after acute or chronic lithium administration, as well as after treatment with the cholinergic agonist pilocarpine alone or in combination with lithium at a dose that induces seizures only in lithium pretreated rats. Neither acute nor chronic lithium treatment altered the hippocampal or cortical concentration of Ins 1,4,5P3, cyclic AMP, or cyclic GMP. Pilocarpine administered alone increased Ins 1,4,5P3 in both regions, did not alter cyclic AMP, and slightly increased cyclic GMP in the cortex. Coadministration of lithium plus pilocarpine caused large increases in the concentrations of all three second messengers and the production of each of them was uniquely attenuated: lithium reduced pilocarpine-induced increases of Ins 1,4,5P3 in the cortex at 60 min; chronic lithium administration reduced stimulated cyclic AMP production in the hippocampus; and chronic lithium treatment impaired stimulated cyclic GMP production in both regions. In summary, chronic lithium treatment appeared only to reduce Ins 1,4,5P3 and cyclic AMP concentrations after a long period of stimulation whereas cyclic GMP production was reduced by chronic lithium administration after both short and long periods of stimulation. Thus cyclic GMP was most sensitive to lithium and lithium attenuation of second messenger formation may be most important in excessively activated pathways.     

Effect of aminophylline on seizure thresholds and brain regional cyclic nucleotides in the rat

Male rats were given 150 mg/kg aminophylline i.p. and pentylenetetrazol and maximal electroshock thresholds were determined at 5, 20, 60, and 180 min. Maximal lowering of both thresholds occurred at 20 min postinjection. At that time, markedly elevated cyclic GMP was found in the hippocampus, with lesser elevations in striatum and cortex and no change in thalamus. Cyclic AMP markedly decreased in all regions except the cortex. These findings suggest that aminophylline may lower seizure thresholds by altering the relative concentrations of cyclic nucleotides so that cyclic GMP increases and cyclic AMP decreases. Our experiments do not exclude the possiblity that aminophylline may produce convulsions by specifically blocking the receptor for endogenous adenosine, resulting in increased and sustained neuronal firing.     

In vivo reversal of thyroxine induction of DNA synthesis by dibutyrly cyclic AMP in developing rat cerebellum.

Thyroxine, dibutyryl cyclic AMP, and a combination of both drugs were administered daily from birth to 2, 2 and 3 pups, respectively from each of 5 litters of Sprague-Dawley rats. Body weight, brain weight, cerebellar weight, and cerebellar DNA were measured in each animal at age 5 days and compared with values from a pair of controls from each litter. Cerebellar weight and DNA content were affected more severely than body weight in cyclic AMP-treated animals, with cerebellar DNA reduced significantly to 88% of control values. Cerebellar DNA was significantly elevated to 117% of control values in thyroxine-treated animals. This augmentation of cerebellar DNA synthesis by thyroxine was negated by administration of dibutyryl cyclic AMP 10 min prior to the thyroxine injection. These results support an hypothesis that the enhancement of cerebellar cell division by thyroxine involves an increase in the ratio of intracellular cyclic guanosine monophosphate to cyclic adenosine monophosphate. The reversal of the thyroxine-induced increase in cerebellar DNA synthesis by a prior injection of dibutyryl cyclic AMP suggests that the early stimulation of cell division by thyroxine may be mediated by cyclic AMP, and that the intracellular balance between cerebellar cyclic AMP and cyclic GMP was distorted by in vivo elevation of intracellular cyclic AMP levels.     

Cyclic adenosine monophosphate in cerebral cortex. Alterations following trauma.

Stab-wound injury produced a seven-fold elevation in cyclic adenosine monophosphate (AMP) in mouse brain within one minute. The increase in cyclic AMP in the brain was blocked by prior treatment of the animal by theophylline, chlorpromazine, trifluoperazine hydrochloride, and diphenhydramine hydrochloride. Neither dichloroisoproterenol, pronetalol, nor reserpine blocked in the rise in cyclic AMP concentration due to injury. The results following administration of drugs suggest that the increases in cyclic AMP due to injury may be mediated by adenosine. Theophylline and phenothiazine derivatives have been shown previously to decrease adenosine-mediated increases in cyclic AMP in brain slices. The absence of any effect after administration of dichloroisoproterenol or pronetalol suggests that the increase in cyclic AMP after injury is not through catecholamine release. Hypothermia reduced the increase in cyclic AMP in injured brain after one minute.     

Cyclic nucleotides and seizures in a hereditary model of epilepsy

The high seizure susceptibility in epileptic fowl is due to an autosomal recessive mutation. Cyclic AMP and cyclic GMP concentrations were determined in brains from two day old epileptic chicks (homozygotes) during an inter-ictal period as well as during and following a seizure evoked by stroboscopic stimulation. The data were compared to values obtained from non-epileptic carrier chicks (heterozygotes) sacrificed in an unstimulated state or subjected to the seizure evoking stimulus. During the inter-ictal state in epileptics no abnormalities were found in cyclic nucleotide concentrations indicating that the high seizure susceptibility is not related to abnormalities of these nucleotides. Although seizure activity in epileptics was associated with reduced cyclic AMP in the optic lobes this also occurred in carrier chicks subjected to the seizure evoking stimulus. The only significant changes in cyclic GMP levels, occurring as a result of seizures in epileptics, were an increase in cyclic GMP in the cerebral hemispheres during the seizure and a decrease in the optic lobes during the postictal period.     

Inhibitory effects of anticonvulsant drugs on cyclic nucleotide accumulation in brain.

Veratridine causes deplorization of excitable cells and produces marked elevation of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) levels in incubated slices of mouse cerebral cortex. Phenytoin, carbamazepine, phenobarbital, primidone, phensuximide, methsuximide, alpha-methyl-alpha-phenylsuccinimide, and high concentrations of clonazepam are anticonvulsant drugs that preferentially prevent maximal electroshock seizures (MES) and generalized tonic-clonic convulsions; all these agents inhibit veratridine-induced accumulation of both cyclic AMP and cyclic GMP. In contrast, ethosuximide, trimethadione, valproic acid, and low concentrations of clonazepam are anticonvulsant drugs that act predominantly against Metrazol and absence seizures; these agents are ineffective or inhibit accumulation of only cyclic GMP. The results suggest that inhibition of cyclic AMP and cyclic GMP accumulation in depolarized brain tissue is a molecular neuropharmacological action characteristic of anticonvulsant drugs that have direct effects on cellular membrane function and prevent MES. Anticonvulsant drugs that do not inhibit accumulation of both cyclic AMP and cyclic GMP in depolarized brain tissue preferentially prevent Metrazol and absence seizures and probably exert their effects by altering neurotransmission mechanisms.     

Lithium effects on noradrenergic-linked adenylate cyclase activity in intact rat brain: an in vivo microdialysis study.

The effects of chronic lithium treatment on adenylate cyclase activity in intact rat brain were examined using in vivo microdialysis. Basal extracellular cyclic adenosine monophosphate (AMP) increased in a dose-dependent manner after norepinephrine was added to the perfusate. Chronic lithium treatment increased basal brain extracellular fluid cyclic AMP levels, while decreasing the magnitude of the cyclic AMP response to stimulation with 100 microM norepinephrine.     

Effect of cyclic AMP on ammonia-induced alterations in primary astrocyte cultures

Current evidence suggests that astrocytes may be the target of ammonia toxicity. Consistent with this view are recent investigations which have shown morphologic alterations in primary astrocyte cultures following exposure to ammonia. In the present study, these alterations became severely aggravated when the cultures were not grown or maintained in dibutyryl cyclic adenosine monophosphate (AMP). Cyclic AMP analogues and agents that increase intracellular cyclic AMP levels significantly inhibited the toxic effects of ammonia. The exact mechanism responsible for this apparent protective effect of cyclic AMP on ammonia-treated astrocytes is not known. The possible means by which cyclic AMP may serve to ameliorate ammonia-induced toxicity are discussed.     

The role of cyclic nucleotides in the CNS.

On the basis of the information presented in this review, it is difficult to reach any firm decision regarding the role of cyclic AMP (or cyclic GMP) in synaptic transmission in the brain. While it is clear that cyclic nucleotide levels can be altered by the exposure of neural tissues to various neurotransmitters, it would be premature to claim that these nucleotides are, or are not, essential to the transmission process in the pre-or post-synaptic components of the synapse. In future experiments with cyclic AMP it will be necessary to consider more critically whether the extracellularly applied nucleotide merely provides a source of adenosine and is thus activating an extracellularly located adenosine receptor, or whether it is actually reaching the hypothetical sites at which it might act as a second messenger. The application of cyclic AMP by intrcellular injection techniques should minimize this particular problem, although possibly at the expense of new diffulties. Prio blockade of the adenosine receptor with agents such as theophylline or adenine xylofuranoside may also assist in the categorization of responses to extracellularly applied cyclic AMP as being a result either of activation of the adenosine receptor or of some other mechanism. Utimately, the developement of highly specific inhibitor for adenylate cyclase should provide a firm basis from which to draw conclusions about the role of cyclic AMP in synaptic transmission. Similar considerations apply to the action of cyclic GMP and the role of its synthesizing enzyme, guanylate cyclase. The use of phosphodiesterase inhibitors in studies on cyclic nucleotides must also be approached with caution. The diverse actions of many of these compounds, which include calcium mobilization and block of adenosine uptake, could account for many of the results that have been reported in the literature.     

Studies on AMP deaminase and 5'-nucleotidase in rat brain under different experimental conditions

Adenosine monophosphate (AMP) deaminase and 5'-nucleotidase, the two enzymes involved in the disposal of AMP, have been detected in different regions of normal rat brain and in animals subjected to heightened neuronal activity (leptazol-induced convulsions) and to depression of the central nervous system (CNS) by the administration of barbiturates. They have also been estimated in the CNS of animals subjected to anoxia or treated with lithium and ammonium salts. The AMP deaminase activity was found to be highest in cerebellum and lowest in cerebral cortex, while the 5'-nucleotidase activity was found to be highest in brain stem and lowest in cerebellum. The AMP deaminase activity was elevated in all the regions of brain during the preconvulsive and convulsive periods. The activity returned to normal during recovery. The activity of 5'-nucleotidase was found to be depressed in the preconvulsive and post-convulsive periods. The enzyme was also found to be depressed in all the three regions after the administration of barbiturates. Administration of lithium or ammonium salts of induction of anoxic states resulted in an increase in the activity of AMP deaminase in all the three regions of brain. These results are discussed in relation to the probable production of cyclic AMP and cyclic guanosine monophosphate (GMP) which may have depressive and excitatory roles, respectively, in brain. It appears that increased AMP deaminase activity is associated with increased neuronal activity while depression of 5'-nucleotidase activity is associated with conditions of decreased CNS excitability.     

Amygdaloid kindling and cerebrospinal cyclic nucleotides

The kindling model of experimental epilepsy is characterized by a persistent seizure pattern and long-lasting seizure susceptibility without associated tissue damage. In order to examine the relationship between CSF cyclic nucleotides and epilepsy. CSF cAMP and cGMP were measured before and after kindling, or after electrically induced seizures. Cyclic AMP and cGMP levels in cisternal CSF decreased significantly 1 week after the amygdaloid kindling. This finding suggests decreased levels of brain cAMP and cGMP in this type of epileptogenesis. A slight increase in CSF cyclic nucleotides concentrations was found after triggering both partial and generalized seizures. There was, however, no difference in increase of cAMP and cGMP levels between partial seizure and generalized convulsion, indicating that differences in intensity ictal or postictal events cannot be reflected in the CSF cyclic nucleotide concentrations.     

Changes in nucleotide hydrolysis in rat blood serum induced by pentylenetetrazol-kindling

There is growing pharmacological evidence from several animal models of seizure disorders that adenosine possesses endogenous anticonvulsant activity. Apart from being released from cells, adenosine can be produced by the degradation of adenine nucleotides by ectoenzymes or soluble nucleotidases. These enzymes constitute an important mechanism in synaptic modulation, as they hydrolyze ATP, an excitatory neurotransmitter, to adenosine, a neuroprotective compound. We recently demonstrated an increase in ectoenzyme activity in rat brain synaptosomes after pentylenetetrazol-kindling in rats resistant to kindling, suggesting a role for ectonucleotidases in the seizure control. The present work investigates the effect of seizures induced by pentylenetetrazol kindling on the enzymes that could be playing a role in ATP, ADP and AMP hydrolysis to adenosine in rat blood serum. Animals received injections of PTZ (30 mg/kg, i.p., dissolved in 0.9% saline) once every 48 h, totaling 10 stimulations and the controls animals were injected with saline. The hydrolysis of ATP, ADP and AMP were significantly increased (42, 40, and 45%, respectively), while phosphodiesterase activity was unchanged. These results suggest once more that an increase in the ATP diphosphohydrolase and 5'-nucleotidase activities and, possibly, in adenosine levels, could represent an important compensatory mechanism in the development of chronic epilepsy. Moreover, the fact that this increase can also be measured in serum could mean that these enzymes might be useful as plasma markers of seizures in epilepsy.     

Phenytoin: effects on calcium flux and cyclic nucleotides.

Previous studies have demonstrated that phenytoin alters calcium conductance in isolated presynaptic nerve endings (synaptosomes) from rat or rabbit brain. Drug concentrations of 0.08 mM (20 microgram/ml) or higher inhibit stimulated calcium influx into synaptosomes depolarized by high concentrations of potassium (69 mM) by 7-58%. Calcium transport into undepolarized synaptosomes is only inhibited by 0.4 mM or greater concentrations of phenytoin. Recent investigations show that in mouse brain slices, phenytoin inhibited elevations of cyclic GMP and cyclic AMP produced by ouabain or veratridine. In contrast, elevations of the two cyclic nucleotides produced by high concentrations of potassium were not inhibited by phenytoin, suggesting that the anticonvulsant suppresses depolarization-induced elevation of cyclic nucleotide levels in brain slices by inhibiting influx of sodium into cells. These data indicate that phenytoin inhibits both sodium and calcium influx into cells during cellular depolarization and alters regulation of brain cyclic nucleotide levels. Both of these actions may be important for the antiepileptic effect of phenytoin.     

Development of adenosine-dependent cyclic AMP accumulation in the avian optic tectum

The present work shows the existence of adenosine-dependent cyclic adenosine monophosphate (AMP) accumulation in the chick optic tectum. When tecta from 18-day-old embryos were incubated with the phosphodiesterase inhibitor IBMX and RO 20-1724, the cyclic AMP level increased from 39.2 to 73.3 and 285.5 pmol/mg protein, respectively. The high level obtained with RO 20-1724 could be inhibited by increasing concentrations of IBMX or by adenosine deaminase, but not by dipyridamole. 2-Chloroadenosine promoted a dose-dependent cyclic AMP accumulation in tecta incubated with RO 20-1724 and adenosine deaminase. This effect was blocked by IBMX and varied substantially during the development of the tissue. The degree of stimulation increased after day 11 of incubation, attaining maximal levels on day 14. The effect of 2-chloroadenosine remained constant until day 18, a period when both the protein content and the basal cyclic AMP levels are increasing in the developing tectum. The cyclic AMP increase elicited by 2-chloroadenosine was greatly reduced in tecta from 20-day-old embryos and 2-day-old chicks. The putative transmitters glutamate and glycine and the neurotransmitter analogs isoproterenol and carbachol had no stimulatory effect on the cyclic AMP accumulation of tecta from 10- and 17-day-old embryos.     

Cyclic AMP levels in cerebrospinal fluid in manic-melancholic patients.

The concentrations of adenosine 3'5'-cyclic monophosphate (cyclic AMP) in cerebrospinal fluid (CSF) in manic-melancholic patients were studied. As control groups served patients suffering from other psychiatric disorders as well as neruological and orthopedic patients. The results showed no difference between the various diagnostic groups, including melancholic versus manic patients, unipolar versus bipolar types. The severity of the affective states measured by rating scales showed no correlation to cyclic AMP levels in CSF. The cyclic AMP levels were apparently not influenced by electroconvulsive therapy or treatment with lithium, neuroleptica, or tricyclic antidepressants.     

Receptors, adenylate cyclase, depression, and lithium

Although numerous studies have suggested that depression may be associated with a reduction in synaptic noradrenaline in the brain, direct beta-adrenergic receptor agonists have not been tested in the treatment of depression until recently. Moreover, newer theories of antidepressant action suggest that a reduction in beta-adrenergic receptor sensitivity is a better correlate of antidepressant treatment than noradrenaline turnover changes. It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta-2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. Plasma cyclic AMP before treatment with salbutamol rose 26% in response to salbutamol 0.25 mg iv. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to iv salbutamol declined over 60%. The beta-adrenergic adenylate cyclase remained subsensitive 4 days after cessation of salbutamol therapy. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. The plasma cyclic GMP response to subcutaneous epinephrine, suggested as a model for presynaptic alpha-noradrenergic mechanisms, is also partially inhibited by Li therapy. Since cyclic AMP and cyclic GMP may be viewed as balancing substances, their interaction may provide a mechanism for Li's dual clinical effects in mania and depression. It is important that in vivo techniques be developed for evaluating receptor changes. The plasma cyclic AMP response to adrenergic stimulation provides an in vivo measure of receptor function that can be useful in studying drug effects during the clinical treatment of humans.     

Correlations between cerebellar cyclic GMP and motor effects induced by deltamethrin: independence of olivo-cerebellar tract

The latencies and incidence of deltamethrin-induced motor symptoms were compared in rats pretreated with saline or 3-acetylpyridine (3-AP) (75 mg/kg i.p. 4 days before). 3-AP produced considerable degeneration of the inferior olive and hence the climbing fiber afferents to the cerebellar cortex. The times of onset of whole body tremor and spontaneous choreiform episodes were accelerated significantly in lesioned rats. Therefore the olivocerebellar tract is not important in mediating deltamethrin symptoms, but is necessary to compensate for the disruption of motor activity and coordination induced by deltamethrin at another site. Cerebellar cyclic guanosine monophosphate (cyclic GMP) levels were elevated significantly during the deltamethrin syndrome. The increase in cyclic GMP levels correlated directly with the duration of motor symptoms. This suggests that deltamethrin has an indirect effect on cerebellar cyclic GMP levels. Lesion of the olive-cerebellar tract did not abolish this rise in cerebellar cyclic GMP levels. The sequential development of the motor symptoms, whole body tremor followed by choreiform episodes was found to have an additive effect in raising cerebellar cyclic GMP levels. This suggests that both the nature and the general level of motor activity can influence cerebellar cyclic GMP levels.     

Calcitonin gene-related peptide (CGRP)-induced cyclic AMP, cyclic GMP and vasorelaxant responses in rat thoracic aorta are antagonized by blockers of endothelium-derived relaxant factor (EDRF).

The mechanism of CGRP-induced vasodilation in rat thoracic aorta was investigated using antagonists of the classical endothelium-derived vasorelaxant factor (EDRF) and comparisons with acetylcholine-induced vasodilations. The CGRP-induced relaxations of isolated rings of rat thoracic aorta were completely dependent on the presence of endothelium and were associated with increases in the levels of both cyclic AMP and cyclic GMP, the same as in our previous study using rat abdominal aorta. Maximum relaxations to CGRP, which represented 40-50% reversal of the norepinephrine-induced contractions, occurred with 100 nM CGRP. Addition of acetylcholine (ACh, 1 microM) to aortic rings, which were already maximally relaxed to CGRP, caused further relaxation to 100%, suggesting that CGRP may use a mechanism (or pool of EDRF) different from that of ACh. Both CGRP- and ACh-induced relaxations of aorta were significantly inhibited by the EDRF blocking agents, hemoglobin (10 microM), methylene blue (10 microM), and nordihydroguaiaretic acid (NDGA, 10 microM). In fact, hemoglobin and NDGA were more effective as inhibitors of CGRP-induced relaxations than ACh-induced relaxations. Hemoglobin, methylene blue and NDGA also inhibited the CGRP-induced increases in both cyclic AMP and cyclic GMP levels. On the other hand, indomethacin, a cyclo-oxygenase inhibitor, did not alter CGRP-induced vasorelaxations or increases in either cyclic AMP or cyclic GMP levels, suggesting that prostaglandins are not involved. Therefore, CGRP-induced vasodilations in rat thoracic aorta appear to involve EDRF, leading to cyclic GMP elevations in smooth muscle and ultimately vasorelaxations. However, another previously undescribed mechanism, which involves EDRF-dependent and indomethacin-resistant elevations of cyclic AMP levels, is triggered by CGRP in thoracic aorta. This novel EDRF-dependent cyclic AMP response may contribute to the CGRP-induced vasodilation in rat thoracic aorta.