Formation and utilization of the active sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate in brain slices: effects of depolarizing agents.

The accumulation and utilization of [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [35S]sulfate. [35S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [35S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [35S]PAPS formation was also indirectly followed by incubating slices in the presence of beta-naphthol and measuring the levels of [35S]beta-naphthyl sulfate ([35S]beta-NS). Whereas [35S]PAPS levels rapidly reached a plateau, [35S]beta-NS formation proceeded linearly with time for at least 1 h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [35S]PAPS formation in slices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC50 = 10 microM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [35S]PAPS accumulation and [35S]beta-NS formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N-methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized.