Multiple forms of cyclic 3′, 5′-AMP phosphodiesterase of rat cerebrum and cloned astrocytoma and neuroblastoma cells

Abstract

The soluble supernatant fraction of rat cerebrum and cloned rat astrocytoma (C6 and C2 1) and mouse neuroblastoma (N1E and N18) cells was subjected to electrophoresis on a preparative polyacrylamide gel column, and the eluted material was analyzed for cyclic AMP phosphodiesterase activity. The cerebrum had four peaks of phosphodiesterase activity, designated I-IV according to the order in which they emerged from the column. The ratio of the activities of these peaks was approximately 1:400:150:200. The C6 astrocytoma cell line had three peaks of phosphodiesterase activity corresponding to Peaks I, II and III of rat cerebrum. The ratio of activities in this cell line was about 1:2:3. The C2 1 astrocytoma cell line had Peaks I and IV only (ratio = 1:1), and both neuroblastoma cell lines had Peak III only. Besides having different electrophoretic mobilities, the different forms of phosphodiesterase had several other distinguishing characteristics. For example, an endogenous, heat-stable activator of phosphodiesterase increased the activity of Peak II up to 10-fold but failed to increase the activity of Peaks I, III and IV. The phosphodiesterase inhibitor, trifluoperazine, inhibited Peak II to a greater extent than it did Peak III, whereas theophylline inhibited Peak III more than Peak II. The peaks also had different stabilities; Peaks III and IV had half-lives of less than 1 day when stored at 4°C. In contrast, Peaks I and II retained most of their original activity even after two weeks of storage. The addition of 1% albumin stabilized all of the enzyme preparations. The crude supernatant fractions of tissues with multiple forms of phosphodiesterase, as evidenced by gel electrophoresis also showed multiple forms of the enzyme by kinetic analysis. On the other hand, the supernatant fraction of neuroblastoma cells evidenced only a single form of the enzyme by gel electrophoresis and exhibited linear kinetics. All the electrophoretically separated peaks of phosphodiesterase also showed linear kinetics. Peaks of phosphodiesterase activity that occurred in a given fraction of the electrophoresis eluant exhibited similar characteristics regardless of the tissue source. For example. Peak II was always activated by the heat-stable activator in all tissue sources. Moreover, theophylline and trifluoperazine, which produced a differential inhibition of the various peaks of phosphodiesterase, inhibited each corresponding peak to the same degree regardless of the source of the enzymes. We conclude that there are multiple forms of cyclic nucleotide phosphodiesterase in the central nervous system and propose that each type of cell may have a unique and definite number and pattern of these forms of the enzyme.