Les P. Davies

UPTAKE AND RELEASE OF D‐ AND L‐ASPARTATE BY RAT BRAIN SLICES

D‐Aspartate is accumulated by slices of adult rat cortex by a high affinity uptake which is abolished if the sodium ions in the incubation medium are replaced by choline. A small uptake of D‐aspartate takes place if the sodium ions are replaced by lithium ions. It appears likely that D‐aspartate shares the same transport system with L‐aspartate, and that the uptake of D‐aspartate is into the same osmotically‐sensitive particles as those which accumulate L‐aspartate. D‐Aspartate is released from cerebral cortex slices by raised potassium concentrations, provided calcium is present in the perfusing buffer.

A purinergic component in the anticonvulsant action of carbamazepine

The tricyclic anticonvulsant carbamazepine inhibited the binding of [3H]phenylisopropyladenosine (PIA) to rat brain membranes with a K1 value of 44 microM, which is in the range of therapeutic levels of this drug in brain and serum. Other anticonvulsants and tricyclic antidepressants had little effect on PIA binding. Carbamazepine weakly inhibited adenosine uptake into rat brain slices at 400 microM. An effect on adenosine receptors may be an important component of the anticonvulsant action of carbamazepine.

Interactions of the Anticonvulsant Carbamazepine with Adenosine Receptors. 1. Neurochemical Studies

Summary: At therapeutic concentrations the tricyclic anticonvulsant carbamazepine inhibited the binding of the adenosine analogue [3H]L‐N6‐phenyli‐sopropyladenosine ([3H]PIA) to rat brain membranes (K, =46 μM)in vitro. Carbamazepine interacted much less potently with muscarinic cholinergic, ñ‐adrenergic, ‐γ‐aminobutyric acid, or L‐glutamate binding sites. Carbamazepine was of lower potency (Ki= 112 μM) as an inhibitor of the binding of the putative A2 adenosine agonist [3H]5′‐N‐ethylcarboxamidoadenosine. GTP greatly reduced the potencies of purine agonists, but not antagonists, as inhibitors of [3H]PIA. The potency of carbamazepine, like that of the antagonist theophylline, was not reduced by GTP. Studies on the adenosine‐stimulated adenylate cyclase activity in guinea pig brain slices also revealed theophyllinelike activity of carbamazepine. The possible relevance of agonist and antagonist interactions with adenosine receptors to the anticonvulsant action of carbamazepine is discussed.

POSTNATAL CHANGES IN THE POTASSIUM‐STIMULATED, CALCIUM‐DEPENDENT RELEASE OF RADIOACTIVE GABA AND GLYCINE FROM SLICES OF RAT CENTRAL NERVOUS TISSUE

—Using a simple apparatus designed to perfuse nervous tissue mini‐slices retained on glass fibre filter discs, slices of adult (13 week) rat cerebral cortex and spinal cord were shown to release radioactive GABA and glycine, but not 2‐amino‐isobutyric acid, in response to increased potassium ion concentration of the perfusing medium. A major portion of this potassium‐stimulated release was dependent upon the presence of calcium ions in the perfusing medium. Slices of cerebral cortex and spinal cord from rats of 1 day and 10 days postnatal age showed potassium‐stimulated, calcium‐dependent release of radioactive GABA and glycine respectively. These findings are consistent with other evidence that GABA and glycine are functioning as inhibitory transmitters in rats at least as soon as 1 day after birth.

POSTNATAL CHANGES IN THE HIGH AFFINITY UPTAKE OF GLYCINE and GABA IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— High affinity uptake systems for GABA into slices of cerebral cortex and for glycine into slices of spinal cord have been demonstrated in rats of 1 and 10 days postnatal age and compared with the systems in tissue slices from adult rats. For both systems there was an increase in the maximal rate of uptake of the substrate with development. For glycine uptake there was no significant change in apparent Km during development, whereas there was a four‐fold increase in the apparent Km for GABA uptake. There were some changes with development in the apparent substrate specificity of the two systems suggesting increased specificity with maturation. Bicuculline and strychnine, antagonists of the postsynaptic inhibitory actions of GABA and glycine, produced convulsions in 1‐, 2‐ and 10‐day‐old rats following intraperitoneal injection of doses somewhat lower than those required to convulse adult rats. These findings are consistent with other evidence that glycine and GABA are functioning as inhibitory transmitters at least as soon as 1 day after birth.

Pyrazolo[3, 4-d]pyrimidines as adenosine antagonists

A large number of nitrogen heterocycles structurally related to caffeine and theophylline have been tested for activity as adenosine antagonists. Preliminary screening, utilizing displacement of [3H]N6-phenylisopropyladenosine (PIA) binding to rat brain membranes, identified several pyrazolo[3,4-d]pyrimidines with potential antagonist activity. These were then tested for their ability to antagonize adenosine-stimulated adenylate cyclase of guinea-pig slices and to block adenosine receptors which mediate presynaptic inhibition of transmitter release from cholinergic nerves in guinea-pig ileum. Of several compounds found to have antagonist activity, one of these, 4,6-bis-alpha- carbamoylethylthio -1-phenylpyrazolo[3,4-d]pyrimidine ( DJB -KK) was approximately an order of magnitude more potent than theophylline in both tests. GTP greatly reduces the potency of purine agonists, but not antagonists, as inhibitors of [3H] PIA binding; the potency of the pyrazolo[3,4-d]pyrimidine compounds was not altered by GTP. The compounds have no significant activity against [3H]adenosine uptake or on the binding of ligands to muscarinic cholinergic, beta-adrenergic, GABA or L-glutamate receptors.

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-(3)H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [(3)H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions?. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.

Pyrazolo [3,4-d] pyrimidines, a new class of adenosine antagonists

A variety of nitrogen heterocycles structurally related to caffeine and theophylline have been tested for activity as adenosine receptor antagonists. Preliminary screening, utilizing displacement of [3H]N6-phenylisopropyladenosine binding to rat brain membrane A1-adenosine receptors, identified several pyrazolo[3,4-d]pyrimidines with potential antagonist activity. These were then tested for their ability to antagonize the adenosine-stimulated adenylate cyclase system of guinea-pig brain slices. One of these, 4,6-bis-alpha-carbamoylethylthio-1-phenylpyrazolo[3,4-d]pyrimidine (DJB-KK), was over an order of magnitude more potent than theophylline in blocking adenosine-stimulated increases in cyclic AMP levels.

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2‐OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10‐day‐old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1‐ and 10‐day‐old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2‐oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10‐day‐old rats suggests that the activity of this enzyme reflects cell growth rate.

Syntheses, pharmacological evaluation and molecular modelling of substituted 6-alkoxyimidazo[1,2-b]pyridazines as new ligands for the benzodiazepine receptor

Summary A series of 2,3-disubstituted-6-alkoxyimidazo[1,2-b]pyridazines has been synthesized and evaluated for in vitro affinity for the benzodiazepine receptor (BZR). 3-(Benzamidomethyl or substituted benzamidomethyl)-6-methoxy-2-(3,4-methylenedioxyphenyl)imidazo[1,2-b]pyridazines were found to be the most potent BZR ligands (eg, 4a, IC50 7 nM; 4e, IC50 14 nM; 4v, IC50 8 nM). Imidazo[1,2-b]pyridazines unsubstituted in the 3-position, or containing bulkier alkoxy groups in the 6-position, were found to bind less strongly to the BZR. Selected compounds from the series were identified from in vitro GABA-shift experiments as BZR agonists. Molecular modelling has been employed to identify the common pharmacophoric points of lipophilic and hydrogen bonding, ligand-receptor interaction and areas of steric hindrance for these substituted imidazo[1,2-b]pyridazines at the BZR.