The stable prostacyclin derivative, 7-oxo-prostacyclin, exhibits a delayed, long-lasting cardioprotective effect, which is accompanied by an increase in cyclic nucleotide phosphodiesterase (PDE) activities restricted to the Ca2+-calmodulin-dependent (PDE1) and cyclic AMP-specific phosphodiesterase (PDE4) activities. Mammalian PDEs form a large multi-gene family with differential expression occurring in a cell- and tissue-specific manner. The aim of this study was to identify which isoforms of PDE1 and PDE4 are present in the hearts of control and 7-oxo-prostacyclin treated rats. Using RT-PCR analysis, we were able to identify in control rat hearts transcripts for PDE1C, but not for either PDE1A or PDE1B within the three-gene PDE1 family. Within the four-gene PDE4 family we detected, by generic RT-PCR analysis, transcripts for PDE4A, PDE4B and PDE4D, but not PDE4C. Using RT-PCR primers for specific splice variants, we identified transcripts for PDE4B1, PDE4B2, PDE4B3, PDE4D1, PDE4D2 and PDE4D3 in hearts from the control animals. Immunoblotting of hearts from the control animals for PDE4 forms allowed us to identify a 98-kDa PDE4A species, 68-kDa band representing PDE4D1/2 and a 95-kDa species indicative of PDE4D3. In the hearts of treated animals, 48 h after a single 50 microgram/kg dose of 7-oxo-prostacyclin, a profound increase in transcript levels was seen for both PDE1C and PDE4B3 together with a slight elevation for PDE4B1. No change in PDE4A transcripts occurred, which was consistent with a lack of change indicated in immunoblotting analyses. In contrast, 7-oxo-prostacyclin treatment caused decrease in transcript levels for PDE4D, which was confirmed by immunoblotting and shown to be due to a reduction in the levels of PDE4D3 and also in PDE4D1/D2. Thus, treatment of animals with 7-oxo-prostacyclin initiated profound isoform-specific changes in PDE expression in the myocardium which, presumably, underpin the increased PDE activity.
Copyright 1997 Academic Press Limited.