Rat cerebral-cortex adenosine deaminase activity and its subcellular distribution

Abstract

A radioisotopic assay for adenosine deaminase (EC 3.5.4.4) is described together with its application in investigating the activity of the enzyme in rat cerebral cortex. Activity of the adenosine deaminase was determined to be 115nmol/min per g of tissue, measured in isoosmotic sucrose dispersions of the neocortex, and to be 170nmol/min per g of tissue after treatment with Triton X-100. The enzyme was concluded to be largely cytoplasmic, with a K(m) of 54-57mum for adenosine. Action of the deaminase, and other aspects of the metabolism of adenosine in intact neocortical tissue, were quantitatively appraised on the basis of the newly determined characteristics.

Biochemical characterization of putative central purinergic receptors by using 2-chloro[3H]adenosine, a stable analog of adenosine.

“…Evidence for depolarization-evoked, calcium-dependent release of Ado and ATP from brain slices (18,19) and synaptosomal preparations (12, 20) suggests a potential physiological role for purine-modulated alterations in cell firing, transmitter release and cyclic AMP metabolism, and their sequelae.…”

“…Conversely, theophylline and other adenosine antagonists increase cell firing (11). Although the effects of the purines on cell firing may be attributed to inhibition of excitatory neurotransmitter release, it is thought that a direct inhibitory effect via purinergic PI receptors may also occur; however, there is controversy about the evidence for the existence of P2 receptors in the central nervous system (3).Behaviorally, Ado has sedative, hypnotic, and anticonvulsant activity (13-16), in contrast to caffeine and theophylline, which are central stimulants (17).Evidence for depolarization-evoked, calcium-dependent release of Ado and ATP from brain slices (18,19) and synaptosomal preparations (12, 20) suggests a potential physiological role for purine-modulated alterations in cell firing, transmitter release and cyclic AMP metabolism, and their sequelae.Demonstration of high-affinity binding of a suitable radioligand to brain membranes has become an additional criterion for the identification of an endogenous substance as a putative neurotransmitter. For Ado and ATP, their involvement in nearly all facets of cell function (21) Subcellular fractions from rat brain were prepared by the method of Whittaker (27); the nuclear, crude synaptosomal, myelin, enriched synaptosomal, mitochondrial, and microsomal subfractions were homogenized by using a Polytron (Brinkmann PT 20-ST generator, setting 5.5 for 10 sec) in distilled water; centrifuged at 48,000 X g for 10 min; and stored at -80°C for at least 18 hr before use.…”

“…Evidence for depolarization-evoked, calcium-dependent release of Ado and ATP from brain slices (18,19) and synaptosomal preparations (12,20) Purity of the radioligand was checked on a biweekly basis against a reference standard of unlabeled ClAdo by the highperformance liquid chromatography method of Anderson and Murphy (31) and was consistently greater than 99%. Examination of bound radioligand after elution from filters into absolute alcohol and lyophilization showed a single peak of radioactivity that had a k' identical to that of the original radioactive material and unlabeled ClAdo.…”

Inosine, an endogenous ligand of the brain benzodiazepine receptor, antagonizes pentylenetetrazole-evoked seizures.

“…However, the protective effects of adenosine against audiogenic seizures are still manifest after the sedative effects are no longer apparent. In view of the action of ICV administered inosine on pentylenetetrazole-evoked seizures, the anticonvulsant effects of adenosine may result from the deamination of adenosine to inosine, because adenosine deaminase activity in the brain is high (21,22) compared with peripheral tissues. Nevertheless, our findings that inosine and closely related purines have pharmacological effects not unlike those expected of a short-acting benzodiazepine suggest that such purines may function as endogenous ligands for the benzodiazepine receptor.…”

Regional Blood-Brain Barrier Responses to Central Cholinergic Activity

“…Available data support a role for adenosine in the modulation of neuronal activity (Williams, 1984;Dunwiddie, 1985;Phillis and Wu, 1983;Fredholm and Hedqvist, 1980). Adenosine and its receptors have wide-spread distribution in the nervous system, and receptor activation is associated with both presynaptic and postsynaptic inhibitory effects (Kuroda and McIlwain, 1977;Stone, 1981;Bender et al, 1981;Pull and McIlwain, 1974. A variety of seizure models indicate that stable adenosine analogs have potent anticonvulsant effects (Albertson et al (1983); Rosen and Berman, 1985;Dragunow and Goddard, 1985;Maitre et al, 1974;Dunwiddie and Worth, 1982;Murray et al, 1985;Burley and Ferrendelli, 1984;Turski et al, 1985).…”