An Improved, Automated Adenylate Cyclase Assay Utilizing Preparative HPLC: Effects of Phosphodiesterase Inhibitors

Abstract

Analysis of adenylate cyclase (ACase) activity in broken cell preparations usually involves conversion of [α-³²P]ATP to [³²P]cyclic AMP (cAMP) followed by purification of cAMP by liquid chromatographic methods. An automated, preparative reverse-phase HPLC procedure was developed that purifies cAMP rapidly and decreases variability and background. It permits the separation procedure to be validated rapidly prior to use with actual samples, and is readily adaptable for assaying guanylate cyclase, phosphodiesterases (PDE), or a variety of other related nucleotide-metabolizing enzymes. For ACase assays, 4.5% ZnSO₄-10% Ba(OH)₂ is added to the incubation mixture, and following centrifugation, the supernatant is injected on an HPLC apparatus fitted with a Waters Z-Module containing a 10-μ C₁₈ reverse-phase cartridge. Using a mobile phase of 0.15 M sodium acetate-20% methanol (pH 5.0) at a flow rate of 4 ml/min, cAMP is eluted at k′ > 1.25, whereas k′ < 0.5 for all other adenine nucleotides, permitting collection of the cAMP fraction after running the other nucleotides to waste. The method was validated by characterizing dopamine-sensitive ACase in homogenates of striatum from Sprague-Dawley rats. Basal activity (177 ± 16 pmol/mg protein/min), the stimulation by dopamine (186 ± 19 pmol/mg/min), the apparent K_m for dopamine (5.0 ± 1.5 μM), and expected effects of varying magnesium, EGTA, and GTP were similar to available data. However, it was found that isobutylmethylxanthine (IBMX) or theophylline, usually included in the incubation mixture as PDE inhibitors, markedly inhibited the synthesis of cAMP in both the presence and absence of dopamine. A consequence of this inhibition was a marked change in the apparent K_m of dopamine calculated from a Lineweaver-Burk plot. The use of IBMX to inhibit PDEs was compared with an alternate strategy, the addition of excess exogenous cAMP. Simultaneous analysis of PDE and ACase activity was accomplished by including [³H]cAMP in the incubation and quantifying the amounts of [³H]cAMP hydrolyzed and [³²P]cAMP synthesized. Without IBMX, a concentration of 1 mM exogenous cAMP was sufficient to prevent significant loss of [³H]cAMP. In the absence of exogenous cAMP, 0.5 mM IBMX did not completely prevent the breakdown of [³H]cAMP, whereas 2.5 mM IBMX did. Although there was 25% less [³H]cAMP recovered in the presence of 0.5 mM IBMX than with 2.5 mM IBMX, there was no difference in the amount of [³²P]cAMP formed (either with or without dopamine). Moreover, in the presence of IBMX, there was a 20–30% lower synthesis of [³²P]cAMP compared with incubations in which only 1 mM cAMP was used to prevent breakdown of [³²P]cAMP. These data suggest that alkylxanthines, possibly through effects on adenosine receptors, may cause unexpected effects on estimations of dopamine-stimulated ACase. The use of exogenous cAMP as an alternate substrate for PDEs may be one way to obviate these problems.