Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes, from liver and hepatocytes, identifies a novel IBMX-insensitive form

Abstract

DEAE chromatography of a high speed supernatant fraction from a homogenate of rat liver, prepared under isotonic conditions in the presence of protease inhibitors, yielded three peaks of cyclic nucleotide phosphodiesterase activity (PDE activity). The first peak could be resolved on Affi-gel Blue chromatography to yield a Ca 2+/calmodulin stimulated cyclic GMP specific PDE and a cyclic AMP and cyclic GMP hydrolysing PDE whose activity was insensitive to Ca 2+/calmodulin. These two activities could also be clearly resolved by Mono-Q chromatography of soluble extracts from both liver and hepatocytes. These had different molecular weights, kinetics of substrate utilization, thermostabilities, dependence on Mg 2+ and inhibitor sensitivities. The cyclic AMP and cyclic GMP utilizing PDE resolved in these procedures appears to be a novel enzyme form (PDE-MQ-I) which is insensitive to inhibition by the so-called non-selective PDE inhibitor IBMX and displays catalytic activity in the absence of Mg 2+. None of the inhibitors tested were capable of inhibiting this form showing that the catalytic activity of this species could be distinguished from all the other soluble activities. This novel enzyme hydrolysed both cyclic AMP and cyclic GMP with K m values of 25 μM and 237 μM, respectively. The V max ratio of hydrolysis of cyclic GMP/cyclic AMP was above unity (1.4). It accounted for 30% of the soluble cyclic AMP PDE activity and 10% of the cyclic GMP PDE activity assessed at 1 μM substrate. Gel filtration of PDE-MQ-I indicated a size of 33,150 Da, in contrast to the size of 237,500 Da observed for the Ca 2+/calmodulin PDE-MQ-II. Thermal inactivation of PDE-MQ-I and PDE-MQ-II yielded single exponential decays with t 1 2 values of 6.33 min and 0.7 min at 60° respectively. In the presence of saturating Ca 2+, PDE-MQ-II was activated by calmodulin with an EC 50 of ca. 30 ng/ml. In the presence of calmodulin, PDE-MQ-II was activated by Ca 2+ with an EC 50 of ca. 20 μM. Chromatography of homogenates on Mono-Q also identified a cyclic GMP-activated cyclic nucleotide PDE (PDE-MQ-III) and two cyclic AMP specific activities (PDE-MQ-IV and PDE-MQ-V). These exhibited very different inhibitor sensitivities and could be readily distinguished using the compound Ro-20-1724 which yielded IC 50 values for inhibition of >500 μM, 13 μM and 1.5 μM, respectively, for the hepatocyte enzymes. PDE-MQ-III to -V exhibited K m values for the hydrolysis of cyclic AMP of 37.7 μM, 0.62 μM and 0.62 μM and Hill coefficients of 1.62, 0.69 and 1.01, respectively. Rolipram was found to potently inhibit both the cyclic AMP specific forms. The compound ICI 118233 failed to inhibit any of the isolated soluble phosphodiesterases. A similar multiplicity of phosphodiesterase species was observed in the soluble fraction from both rat liver homogenates and isolated hepatocytes. We suggest that care be taken in interpreting experiments performed to assess adenylate cyclase activity in intact cells by monitoring intracellular cyclic AMP accumulation in the presence of IBMX due to the possible presence of an IBMX-insensitive phosphodiesterase.