Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei

The identity and location of nuclear cyclic nucleotide phosphodiestsrases ( PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fracti ons from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensiti ve to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the ca rdiac nuclear envelopes (NE) were similar to those measured in the correspo nding intact nuclei, thus suggesting that most PDE activity is associated w ith the nuclear membrane. Moreover, the main hydrolytic activities in cardi ac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC50, K-m and K-i values was typic al of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-sti mulated PDE) represented a minor component (8-9%) of total hydrolytic activ ity. Solubilization of nuclear envelopes and HPLC separation also yielded r olipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the pre sence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enri ched, P1 pellet. In addition, Western blot analysis demonstrated the presen ce of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. H owever, in the same preparations, the presence of PDE4A could not be ascert ained. Altogether, these results suggest an intrinsic and predominant assoc iation of these nuclear PDEs with the NE and much likely with nucleopore co mplexes. (C) 1999 Elsevier Science B.V. All rights reserved.