1. Uptake of [(14)C]adenine and [(14)C]adenosine from surrounding fluids to guinea-pig cerebral tissues was measured during incubation in vitro. Output of (14)C-labelled compounds from the loaded tissues to superfusion fluids occurred on continued incubation, at about 0.2% of the tissue's content/min, and this rate was increased about fourfold by electrical excitation of the tissue. 2. The compounds released from the tissue to superfusion fluids included adenine, adenosine, inosine and hypoxanthine with small amounts of nucleotides. Output of all these compounds, except adenine, increased on excitation. Media depleted of oxygen or glucose also increased the output of (14)C-labelled derivatives from [(14)C]adenine-loaded tissues, and this augmented output was further increased by electrical stimulation. 3. [(14)C]Adenosine was found as the main product from [(14)C]ATP when this was added at low concentrations to fluids superfusing cerebral tissue. Metabolic and neurohumoural explanations of the liberation and action of adenosine derivatives in the tissue are discussed.

Adenine nucleotides of guinea-pig neocortical tissues were labelled by incubation with [14C]adenine and excess of adenine was then removed by superfusion with precursor-free medium. Adenine derivatives released from the tissue during continued superfusion, including a period of electrical stimulation of the tissue, were collected by adsorption and examined after elution and concentration. The stimulation greatly increased the 14C output, and material collected during and just after stimulation had a u.v. spectrum which indicated adenosine to be a major component. The additional presence of inosine and hypoxanthine was shown by chromatography and adenosine was identified also by using adenosine deaminase. Total adenine derivatives released from the tissue during a 10min period of stimulation were obtained as hypoxanthine, after deamination and hydrolysis of adenosine and inosine, and amounted to 159nmol/g of tissue. This corresponded to the release of approx. 7pmol/g of tissue per applied stimulus. The hypoxanthine sample derived from superfusate hypoxanthine, inosine and adenosine was of similar specific radioactivity to the sample of inosine separated chromatographically, and each was of higher specific radioactivity than the adenine nucleotides obtained by cold-acid extraction of the tissue.

1. Neocortical tissues, exposed briefly to [(14)C]adenine and containing over 98% of their (14)C as adenine nucleotides, when superfused with glucose-bicarbonate salines released about 0.1% of their (14)C content/min to the superfusate. 2. Addition of unlabelled adenosine to the superfusing fluid increased the (14)C output three- to four-fold; half-maximal increase was given by about 40mum-adenosine, and reasons are adduced for considering the activity of adenosine kinase to be a major factor in conditioning the (14)C output. Adenosine similarly increased the enhanced (14)C output caused by electrical excitation of the superfused tissue; it brought about only a small increase in tissue glycolysis. 3. Output of (14)C from the [(14)C]adenine-labelled tissues was increased when Ca(2+) was omitted from the superfusing fluids, but electrical stimulation did not then liberate more (14)C. Nevertheless, such tissues still responded to electrical stimulation by increased glycolysis, and their (14)C output again became susceptible to increase by electrical stimulation when Ca(2+) was restored. 4. The six-fold increase in tissue glycolysis caused by electrical excitation was almost completely inhibited by tetrodotoxin at 0.1mum and above, but this was associated with about 50% inhibition only in the output of (14)C from tissues preincubated with [(14)C]adenine. The (14)C-labelled compounds of which output was most inhibited by tetrodotoxin were adenosine, inosine and hypoxanthine whereas output in a nucleotide fraction was little affected.

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.

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