Cyclic AMP and cyclic GMP may mediate opposite neuronal responses in the rat cerebral cortex.

Electrophysiologically identified pyramidal tract neurons in the rat cerebral cortex were tested with norepinephrine, acetylcholine, adenosine 3',5'-monophosphate (cyclic AMP), and guanosine 3',5'-monophosphate (cyclic GMP) applied by microiontophoresis. The neurons were usually inhibited by norepinephrine and cyclic AMP, but excited by acetylcholine and cyclic GMP. These opposing responses of pyramidal tract neurons to cyclic AMP and cyclic GMP suggests that these two nucleotides could function as reciprocal intracellular second messengers for norepinephrine and acetylcholine, respectively.

Kynurenines in the CNS: from endogenous obscurity to therapeutic importance.

In just under 20 years the kynurenine family of compounds has developed from a group of obscure metabolites of the essential amino acid tryptophan into a source of intensive research, with postulated roles for quinolinic acid in neurodegenerative disorders, most especially the AIDS-dementia complex and Huntington's disease. One of the kynurenines, kynurenic acid, has become a standard tool for use in the identification of glutamate-releasing synapses, and has been used as the parent for several groups of compounds now being developed as drugs for the treatment of epilepsy and stroke. The kynurenines represent a major success in translating a basic discovery into a source of clinical understanding and therapeutic application, with around 3000 papers published on quinolinic acid or kynurenic acid since the discovery of their effects in 1981 and 1982. This review concentrates on some of the recent work most directly relevant to the understanding and applications of kynurenines in medicine.

Purine metabolism and clinical status of patients with rheumatoid arthritis treated with dipyridamole.

The anti-inflammatory activities of methotrexate and sulphasalazine may be mediated by increases in endogenous adenosine levels. Since the vascular protective drug dipyridamole inhibits the uptake and metabolism of adenosine we have now tested this compound in patients with rheumatoid arthritis to assess its effects on their symptoms. Forty patients (aged 18-75 years) received dipyridamole 400 mg/day or placebo. The levels of adenosine and its major metabolites were determined by high performance liquid chromatography (HPLC) in blood samples taken at baseline and at monthly intervals during treatment for 6 months. After three months of treatment there was a significant reduction in the modified Health Assessment Questionnaire (mHAQ) score, but these effects were not maintained, and dipyridamole did not modify disease severity scores or the levels of adenosine and its metabolites. We conclude that the symptoms of rheumatoid arthritis were not modified by treatment with dipyridamole.

Development and therapeutic potential of kynurenic acid and kynurenine derivatives for neuroprotection.

Manipulation of the kynurenine pathway of tryptophan metabolism has yielded a plethora of agents that are now being developed as neuroprotectants and anticonvulsants. This pathway is involved in the production of the excitotoxin quinolinic acid and the neuroprotectant kynurenic acid. Approaches used in the development of therapeutic agents include production of analogues or pro-drugs of kynurenic acid and inhibitors of the enzyme responsible for the synthesis of quinolinic acid. Indeed, analogues of the amino acid receptor antagonist kynurenic acid are now in, or are about to enter, clinical trials for stroke and related disorders. This review summarizes the mechanism of action of these various agents, the development of glutamate receptor antagonists from kynurenic acid and the range of their potential uses in neurology and psychiatry.

Potential role of adenosine antagonist therapy in pathological tremor disorders.

The continuing lack of effective long-term therapies for Parkinson's disease and other disorders in which a primary symptom is involuntary tremor is leading to a search for alternative pharmacological strategies. Adenosine is a major modulator of neuronal activity and neurotransmitter release in the central nervous system, with A1 receptors inhibiting transmitter release and A2 receptors generally enhancing release of several transmitter systems relevant to the control of movement. The A2a subtype of receptor is especially concentrated in the neostriatum and is co-localised with D2 receptors for dopamine, the affinity of which are reduced by activation of the A2a population. Antagonists of adenosine, such as theophylline, have been reported to improve the tremor in cases of Parkinson's disease and essential tremor, and the development of better and more selective A2a receptor antagonists may prove of value in these disabling disorders.

Modulation of cytokine release by purine receptors in patients with rheumatoid arthritis.

OBJECTIVE: Since adenosine receptors are known to modulate the release of some inflammatory mediators in control subjects, we have examined the effects of the mixed A1 and A2 adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on basal and lipopolysaccharide (LPS)-induced cytokine release in diluted whole blood cultures from rheumatoid arthritis (RA) patients and healthy volunteers. METHODS: Twenty-eight patients with rheumatoid arthritis aged 18-75 years gave their voluntary consent to participate and give a blood sample. Basal levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) were measured by ELISA, and whole blood cultures were prepared to assess the effects of LPS activation. RESULTS: Following a 40-hour incubation, activation of adenosine receptors by NECA, added to the cell cultures from rheumatoid arthritis patients, was found to suppress both the basal and LPS-induced release of TNF-alpha and IL-1beta, while causing an increase in the release of both basal and LPS-induced IL-6. In healthy volunteers basal cytokines were undetectable, but NECA alone induced the release of all three cytokines. Stimulated levels of TNF-alpha were more than double those in patients. In the control blood cultures, NECA suppressed LPS-induced release of TNF-alpha and IL-1beta, but increased IL-6 release. CONCLUSIONS: Adenosine receptor stimulation has a differential effect on the release of pro-inflammatory cytokines, and may induce cytokine release in normal subjects. Stimulated release of TNF-alpha is substantially lower in patients with rheumatoid arthritis than in control subjects, possibly indicating saturation, exhaustion or down-regulation of the release process.

Altered hippocampal plasticity by prenatal kynurenine administration, kynurenine-3-monoxygenase (KMO) deletion or galantamine.

Glutamate receptors sensitive to N-methyl-D-aspartate (NMDA) are involved in embryonic brain development but their activity may be modulated by the kynurenine pathway of tryptophan metabolism which includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Our previous work has shown that prenatal inhibition of the pathway produces abnormalities of brain development. In the present study kynurenine and probenecid (both 100mg/kg, doses known to increase kynurenic acid levels in the brain) were administered to female Wistar rats on embryonic days E14, E16 and E18 of gestation and the litter was allowed to develop to post-natal day P60. Western blotting revealed no changes in hippocampal expression of several proteins previously found to be altered by inhibition of the kynurenine pathway including the NMDA receptor subunits GluN1, GluN2A and GluN2B, as well as doublecortin, Proliferating Cell Nuclear Antigen (PCNA), sonic hedgehog and unco-ordinated (unc)-5H1 and 5H3. Mice lacking the enzyme kynurenine-3-monoxygenase (KMO) also showed no changes in hippocampal expression of several of these proteins or the 70-kDa and 100-kDa variants of Disrupted in Schizophrenia-1 (DISC1). Electrical excitability of pyramidal neurons in the CA1 region of hippocampal slices was unchanged, as was paired-pulse facilitation and inhibition. Long-term potentiation was decreased in the kynurenine-treated rats and in the KMO(-/-) mice, but galantamine reversed this effect in the presence of nicotinic receptor antagonists, consistent with evidence that it can potentiate glutamate at NMDA receptors. It is concluded that interference with the kynurenine pathway in utero can have lasting effects on brain function of the offspring, implying that the kynurenine pathway is involved in the regulation of early brain development.

Purine suppression of proliferation of Sertoli-like TM4 cells in culture.

The effect of adenosine and related compounds on the proliferation of cultured TM4 cells, a Sertoli-like cell line, has been examined. Adenosine, as well as A1 and A2 adenosine receptor agonists (cyclohexyladenosine and N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine) inhibited cell proliferation. These effects were prevented by 8-cyclopentyl theophylline, 1,3-dimethyl-propargylxanthine and caffeine, antagonists at the A1, A2 and both receptors, respectively. The xanthines had no effect by themselves and, consistent with this, the bathing medium was found not to contain detectable levels of adenosine. It is concluded that TM4 cell proliferation can be regulated by receptors for adenosine.

Presynaptic actions of adenosine are magnesium-dependent.

Reducing the extracellular magnesium concentration of medium bathing a rat hippocampal slice preparation greatly reduced the ability of adenosine to inhibit the synaptically evoked population potential. This loss of potency does not appear to be mediated by increased NMDA receptor activity or increased calcium influx. The results suggest that magnesium is required at the A1 adenosine receptor for its interaction with adenosine.

Increased long-term potentiation in the CA1 region of rat hippocampus via modulation of GTPase signalling or inhibition of Rho kinase.

There is accumulating evidence that Ras, and Ras-related GTPases of the Rho family, such as RhoA, RhoB and Rac1, are involved in synaptic plasticity in brain regions such as the hippocampus. We have recently shown that Rho family GTPases are activated by synaptic transmission in the CA1 region of the hippocampus. Since the function of these GTPases is dependent on post-translational isoprenylation by either farnesyl or geranylgeranyl transferases, we tested the hypothesis that inhibition of isoprenylation would modify long-term potentiation (LTP). Farnesyl transferase inhibition, which suppressed activation of RhoB and Ras but not RhoA or Rac1, reduced the magnitude of LTP, while geranylgeranyl transferase inhibition, which inhibited RhoA and Rac1 but not RhoB, increased the magnitude of LTP. In addition, Y-27632, a specific inhibitor of a downstream effector of Rho GTPases-Rho-kinase-also increased the magnitude of LTP. This provides strong evidence that GTPases are important mediators of synaptic plasticity, and demonstrates that Rho-kinase acts to reduce the degree of plasticity at hippocampal synapses during LTP. Rho-kinase inhibitors have the unusual property of increasing the magnitude of LTP, and so may be potential cognitive enhancers.

Activation of thermogenesis of brown fat in rats by baclofen.

Injection of baclofen (0.5-5 micrograms) into the ventromedial hypothalamus (VMH) of anaesthetized rats produced marked increases in the temperature (over 2 degrees C) and thermogenic activity of brown adipose tissue (BAT). These effects were abolished by ganglionic or beta-adrenergic blockade, or denervation of the tissue, but unaffected by hypophysectomy, adrenalectomy or vagotomy. Injections into the hypothalamus close to, but outside the ventromedial hypothalamus did not affect brown adipose tissue. Intravenous administration of baclofen produced similar increases in the temperature of brown adipose tissue, but at larger doses (50-1000 micrograms), and a subcutaneous injection stimulated the metabolic rate in conscious rats. Chronic treatment with baclofen suppressed weight gain and stimulated activity of brown adipose tissue without affecting food intake. These effects of baclofen, which were not mimicked by injections of gamma-aminobutyric acid (GABA), indicate a novel action of baclofen in the ventromedial hypothalamus, leading to marked increases in metabolic rate and body temperature by stimulating sympathetic outflow to brown fat.

Injection of baclofen into the ventromedial hypothalamus stimulates gastric motility in the rat.

Injection of 2 micrograms (+/-)-baclofen into the ventromedial hypothalamus (VMH) of urethane-anaesthetized rats resulted in an increase in gastric tone and amplitude of contractions. This effect was curtailed by administration of atropine methyl nitrate (20 mg/kg i.p.) or bilateral cervical vagotomy. These results provide evidence for a hypothalamic modulation of gastric motility by the vagus. Baclofen, possibly acting on receptors insensitive to gamma-aminobutyric acid (GABA), may be mimicking a vagal activation system, located within the ventro-medial hypothalamus.

Activity of beta-kainic acid on neocortical neurons in vivo and hippocampal neurons in vitro.

The beta-isomer of kainic acid has been reported to have anticonvulsant activity with a profile suggesting amino acid antagonism. The present study, on neurons in the cerebral cortex of anaesthetised rats or in hippocampal slices, shows that beta-kainate does not antagonise any of the major agonists at amino acid receptors: N-methylaspartate, quisqualic acid or alpha-kainic acid. beta-Kainate does have an excitant action of its own which can be partially reduced by 2-amino-5-phosphonovaleric acid but is unaffected by gamma-D-glutamylglycine. It is blocked completely by kynurenic acid.

Muscimol-induced long-term depression in the hippocampus: lack of dependence on extracellular calcium.

We have recently reported a new protocol for inducing long-term depression through activation of GABAA receptors in the hippocampal site. This long-term depression is reversed by bicuculline and potentiated by neurosteroids such as alphaxalone and 5 alpha-pregnan-3 alpha-ol-20-one. It was also shown that glutamate receptor activity is not involved in the induction of this type of long-term depression. The present study investigates the role of calcium in the induction of this novel form of long-term depression and attempts to determine the mechanism of reversal of muscimol-induced long-term depression. Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum (0.01 Hz). It was observed that the muscimol-induced long-term depression can be obtained in the absence of calcium in the bathing medium. In addition to this, the long-term depression was reversed by N-methyl-D-aspartate, kainic acid, high potassium medium, veratrine and the calcium ionophore A23187 but not high calcium (10 mM) medium. High potassium medium in the absence of calcium reversed the long-term depression induced by muscimol 10 microM. The results suggest that this type of glutamate-independent long-term depression can be induced in the absence of extracellular calcium. Extracellular calcium is not necessary for reversal of the long-term depression, although when intracellular calcium levels are raised, as by A23187, this is capable of inducing reversal.

The effects of adenine dinucleotides on epileptiform activity in the CA3 region of rat hippocampal slices.

Alpha, omega-adenine dinucleotides (Ap(n)A) consist of two adenosine molecules linked at the 5' position by phosphate groups, the number of which is denoted by n and can range from 2 to 6. The aim of this study was to investigate the effect of Ap4A and Ap5A on the rate of epileptiform activity. Hippocampal slices (450 microm), when perfused with a medium containing no added magnesium and 4-aminopyridine (50 microM), generate epileptiform activity of an interictal nature. Ap4A and Ap5A at 1 microM depressed the discharge rate to a significant extent. At this concentration adenosine (1 microM) did not produce any effect. However at 10 microM adenosine, Ap4A and Ap5A all decreased the burst frequency. Adenosine deaminase (0.2 U/ml) totally annulled the inhibition of epileptiform activity produced by 10 microM adenosine or 1 microM Ap4A and Ap5A. Adenosine deaminase did not significantly change the maximum depression of activity produced by 10 microM Ap4A and Ap5A. 8-cyclopentyl-1,3-dimethylxanthine, an A1, receptor antagonist, increased the basal rate of epileptiform activity and prevented the depression of burst discharges by Ap4A. 5'-adenylic acid deaminase converts AMP into IMP which is inactive. 5'-adenylic acid deaminase did not prevent the inhibitory effects of Ap4A. The results suggests that in the CA3 region of the hippocampus, Ap4A and Ap5A act partly by stimulating xanthine-sensitive receptors directly and partly through the formation of the metabolite, adenosine.

Protection against kainate-induced excitotoxicity by adenosine A2A receptor agonists and antagonists.

The neuroprotective role of adenosine receptor agonists in various models of ischaemia and neuronal excitotoxicity has been attributed to adenosine A1 receptor activation. In this study we examine the role of the A2A receptor in the kainate model of excitotoxicity. Kainate (10 mg/kg) was administered systemically 10 min after the intraperitoneal injection of adenosine analogues. The A2A agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680) protected the hippocampus at concentrations of 0.1 and 0.01 mg/kg, but not at 2 microg/kg. The addition of the centrally acting adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine partially reduced protection only in the CA3a region, suggesting that only a small proportion of the protection was attributable to the A1 receptor. A less potent A2A agonist, N6-[2-(3,5-dimethyoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (1 mg/kg), provided only partial protection against kainate. 4-(2-[7-Amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl -amino]ethyl)phenol, a selective A2A antagonist, also showed protection against kainate-induced neuronal death, when administered alone or in combination with CGS21680. These results show that adenosine A2A receptor activation is protective against excitotoxicity. The protection is largely independent of A, receptor activation or blockade.

Extracellular levels of dopamine and its metabolite 3,4-dihydroxy-phenylacetic acid measured by microdialysis in the corpus striatum of conscious AS/AGU mutant rats.

The AS/AGU rat is a mutant derived from the Albino Swiss (AS) strain. It is characterized by an ungainly, staggering gait, hind limb rigidity, whole body tremor and, in older animals, difficulty in initiating movement. As and AS/AGU males aged three, six and nine months (n=6 per group) were used to estimate the levels of dopamine and its metabolites in the extracellular fluid of the caudate-putamen. The results indicate a profound loss of dopamine in the extracellular fluid at all age points examined, together with an increase in the concentration of the metabolite 3,4-dihydroxyphenylacetic acid. It is suggested that these changes reflect a defect of dopaminergic neuron function which may underlie the motor disorder seen in these animals.

Pharmacological analysis of extracellular dopamine and metabolites in the striatum of conscious as/agu rats, mutants with locomotor disorder.

The as/agu rat is a spontaneously occurring mutation which exhibits locomotor abnormalities, reduced tyrosine hydroxylase levels in the substantia nigra and lower extracellular levels of dopamine. The animal could represent a model of some human locomotor disorders. High-potassium medium evoked a 460% rise of dopamine levels in control rats but double this in mutants. Amphetamine increased extracellular dopamine by 710% in controls and 1480% in mutants. Clorgyline produced a small increase of dopamine levels in controls but an 1170% increase in mutants. The uptake inhibitor nomifensine increased dopamine levels by 910% in controls but only 270% in mutants. After treatment with benserazide plus L-DOPA, an acute injection of L-DOPA evoked a release of dopamine which was twice as large in the as/agu rats compared with controls. The results show reduced extracellular dopamine in as/agu rats when the locomotor disorder is apparent, but there has been little loss of tyrosine hydroxylase. The responses to drugs are qualitatively different from those obtained using 6-hydroxydopamine.Overall, the effects of compounds affecting aminergic neurons suggest that one possible mechanism for the neuronal abnormality in as/agu rats is a defective regulation of dopamine release from striatal terminals.

Glutamate as the neurotransmitter of cerebellar granule cells in the rat: electrophysiological evidence.

1 Glutamate and the excitatory aminoacid antagonist, alpha-aminoadipic acid (alphaAA), have been applied by microiontophoresis to Purkinje cells in the rat cerebellum. 2 Glutemate produced excitation of Purkinje cells and alpha AA selectively reduced that excitation without affecting responses to acetylcholine or hydrogen ions. 3 Monosynaptic spikes were evoked in Purkinje cells by stimulating the parallel fibres. alpha AA had little effect on these spikes when applied alone. 4 When the Purkinje cell excitability was reduced by the iontophoresis of gamma-aminobutyric acid, alpha AA then produced railure of the monosynaptic spike on 10 of 13 Purkinje cells, in doses shown to be selectively antagonistic towards aminoacids. 5 These results support neurochemical evidence that glutamic acid may be the neurotransmitter released by granule cell parallel fibres.

A comparison of the effects of morphine, enkephalin, kyotorphin and D-phenylalanine on rat central neurones.

1 Morphine, Met-enkephalin, kyotorphin and D-phenylalanine have been applied by microiontophoresis to neurones in the globus pallidus and cerebral cortex of rats anaesthetized with urethane. 2 In the pallidum, most cells were inhibited by all the agonists, with a high correspondence between cells inhibited by Met-enkephalin and D-phenylalanine and by Met-enkephalin and kyotorphin. Whereas responses to Met-enkephalin were readily antagonized by naloxone, responses to kyotorphin and D-phenylalanine were not. 3 In the cerebral cortex a high proportion of cells was excited by all four agonists and antagonism by naloxone was less consistent than in pallidum. 4 It is concluded that the naloxone-reversible analgesic effects of kyotorphin and D-phenylalanine may be mediated indirectly, rather through an activation of opiate receptors.