¹⁸O labeling of adenine nucleotide α-phosphoryls in platelets: Contribution by phosphodiesterase-catalyzed hydrolysis of cAMP

The kinetics of ¹⁸O incorporation into adenine nucleotide α-phosphoryls was examined in intact human platelets equilibrated with [¹⁸O]water to determine the extent to which this labeling process is related to phosphodiesterase-promoted hydrolysis of cAMP. ¹⁸O incorporation into nucleotide α-phosphoryls under basal conditions could be detected within 1 min and was found to proceed linearly for 20 min at a rate of 70 nmol of ¹⁸O/min/7.8 X 10¹⁰ platelets (equivalent to 1 g wet weight). When platelet cAMP concentration was increased 10- to 40-fold by prostacyclin, the rate of total ¹⁸O incorporation increased 4- to 5-fold during the first 10 min of incubation. Between 10 and 20 min after exposure to prostacyclin, a spontaneous decline in platelet cAMP concentration attributable to 'desensitization' is accompanied by a corresponding decrease in the rate of ¹⁸O labeling of α-phosphoryls. According to ¹⁸O-labeling rates of adenine nucleotide α-phosphoryls, an apparent value of K(m) of 4 to 5 μM and V(max) of approximately 3 nmol/min/mg of protein can be calculated for the composite of operational phosphodiesterase activities in intact platelets. Equating ¹⁸O labeling of adenine nucleotide α-phosphoryls with cellular hydrolysis of cAMP, the t( 1/2 ) for turnover of the entire pool of this cyclic nucleotide is 200 ms under basal conditions and 1.3 s when the basal pool is increased 40-fold by prostacyclin. Exposure of platelets to 1.5 mM 1-methyl-3-isobutylxanthine resulted in over 90% inhibition in the ¹⁸O-labeling rate at 1 min. However, there was a progressive accumulation of cAMP in platelets treated with 1-methyl-3-isobutylxanthine, which was accompanied by a progressive disappearance of inhibition. By 5 min when the new increased steady state level of cAMP was achieved, the rate of ¹⁸O labeling was the same in 1-methyl-3-isobutylxanthine-treated and untreated cells. This transient inhibition by 1-methyl-3-isobutylxanthine is behavior predictable of a dynamic metabolic system that can readjust levels of intermediates to overcome the influence of a competitive inhibitor and maintain metabolite flux. A comparison of the rates and profiles of ¹⁸O incorporation into adenine nucleotide α-, β-, and γ-phosphoryls and orthophosphate indicated that ¹⁸O labeling of nucleotide α-phosphoryls does not derive from the transfer of ¹⁸O-labeled γ-phosphoryl of ATP or orthophosphate. It also revealed a broad potential for this approach to examine multiple aspects of adenine nucleotide metabolism and compartmentation in intact platelets. The ¹⁸O-labeling kinetic data and observed ratios of adenine nucleotide α-phosphoryls containing one, two, or three atoms of ¹⁸O fit a model in which cAMP and related nucleotide metabolites are closely associated with a discrete compartment or complex of metabolic enzymes which prevents their dilution by larger pools of adenine nucleotides during metabolic flux but not the exchange of the nucleotide products with these pools. The results obtained are consistent with ¹⁸O labeling of adenine nucleotide α-phosphoryls deriving primarily or solely from phosphodiesterase-promoted hydrolysis of cAMP in intact platelets. They also indicate that a means of monitoring cyclic nucleotide metabolism and compartmentation in intact cells may be possible by this ¹⁸O-labeling approach.