Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei.
The identity and location of nuclear cyclic nucleotide phosphodiesterases (PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fractions from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensitive to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the cardiac nuclear envelopes (NE) were similar to those measured in the corresponding intact nuclei, thus suggesting that most PDE activity is associated with the nuclear membrane. Moreover, the main hydrolytic activities in cardiac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC(50), K(m) and K(i) values was typical of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-stimulated PDE) represented a minor component (8-9%) of total hydrolytic activity. Solubilization of nuclear envelopes and HPLC separation also yielded rolipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the presence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enriched, P1 pellet. In addition, Western blot analysis demonstrated the presence of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. However, in the same preparations, the presence of PDE4A could not be ascertained. Altogether, these results suggest an intrinsic and predominant association of these nuclear PDEs with the NE and much likely with nucleopore complexes.
Lugnier
C
C
Laboratoire de Pharmacologie et de Physico-chimie des Interactions Cellulaires et Moléculaires, CNRS-UMR, Université Louis Pasteur de Strasbourg, 67401, Illkirch, France.
Keravis
T
T
Le Bec
A
A
Pauvert
O
O
Proteau
S
S
Rousseau
E
E
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006352:Heart Ventricles; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D011719:Pyrazines / Q000494:pharmacology; D020889:Rolipram / Q000494:pharmacology; D012756:Sheep
D007527:Isoenzymes; D011719:Pyrazines; C048350:5-(4-acetamidophenyl)pyrazin-2(1H)-one; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram; D015056:1-Methyl-3-isobutylxanthine