Interaction between adenosine generated endogenously in neocortical tissues, and homocysteine and its thiolactone.

[(14)C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted (14)C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [(14)C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [(14)C]adenine derivatives and diminished the 5?-nucleotides in the tissue to 94% of the acid-extractable [(14)C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide (14)C-derivative when homocysteine was 0.1 mM or greater. The total [(14)C]adenine conversion to non-nucleotide derivatives then increased and the 5?-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.

Colchicine-binding measurements of tubulin in variously sampled and incubated tissues from the mammalian brain.

Tissue preparations from the brain of rats and guinea-pigs were examined for their activity in binding isotopically labelled colchicine, as a measure of their content of tubulin. The amount of binding material extracted into a cold buffered solution was unaffected by keeping tissues at 0 degrees C. It diminished by one quarter when tissues were incubated at 37 degrees C in bicarbonate glucose salines for 1-2 h. This diminution increased when glucose was omitted from incubating solutions and was less when tissues were stimulated electrically. It was modified also by the calcium content of the incubation fluids. Of the binding activity lost on incubation only a little was recovered in surrounding fluids. About half the colchicine-binding activity of the tissues was not extracted by the solution employed; this particulate-attached activity changed little, if at all, on normal incubation but diminished when incubating fluids contained a cyclic AMP-fluoride theophylline mixture which was known to modify tubulin assembly. The retention of both categories of colchicine-binding under normal conditions of incubation is consistent with the ability of such tissues to perform microtubule-dependent processes, notably cytoplasmic transport. Examination of the isolated tissues by the methods reported is of value in indicating which of various factors known to affect separated tubulin and microtubule structures, operate in a cell-containing system under chosen experimental conditions.

Adenosine in cerebral homeostatic role: appraisal through actions of homocysteine, colchicine, and dipyridamole.

Mammalian neocortical tissues were incubated in [14C]adenine-containing fluids and their newly-synthesized adenine derivatives examined after periods of superfusion. Increased [K+] released adenine derivatives from the tissues, a release diminished by homocysteine. Homocysteine acted also to diminish the tissue content of adenosine plus its metabolites hypoxanthine and inosine, while increasing that of S-adenosylhomocysteine. Hypoxia also increased the tissue content and the output of adenosine plus its metabolites, and again homocysteine augmented the S-adenosylhomocysteine. Glutamic acid also increased tissue content and output of adenosine and derivatives, an action diminished by homocysteine and associated with augmented S-adenosylhomocysteine. Colchicine or dipyridamole did not prevent augmentation of S-adenosylhomocysteine by the reagents described; the sequence from adenosine phosphates to S-adenosylhomocysteine is concluded to be intracellular and not to involve extracellular formation of precursor adenosine. Adenosine displayed properties consistent with its being involved in two distinct categories of homeostasis, and also with its exerting an inhibitory tone in normal cerebral systems.