Characterization of the Rolipram-Sensitive, Cyclic AMP-Specific Phosphodiesterases: Identification and Differential Expression of Immunologically Distinct Forms in the Rat Brain

Abstract

To determine the properties of the cAMP-specific, rolipramsensitive phosphodiesterases (cAMP-PDEs) that are expressed in different organs, monoclonal and polyclonal antibodies were raised against different epitopes present in the cAMP-PDE sequences. Of the several antibodies generated against peptides and fusion proteins, one monoclonal and four polyclonal antibodies recognized both the native cAMP-PDEs as well as the denatured proteins on Western immunoblot analysis. An immunoprecipitation assay demonstrated that these antibodies recognized the recombinant rat PDE4A, PDE4B, and PDE4D proteins with different avidity. The polyclonal antibody K118 and the monoclonal M3S1 were most specific for rat PDE4B and PDE4D forms, respectively, whereas the AC55 antiserum displayed the highest affinity for PDE4A forms. This selectivity was confirmed by Western blot analysis using recombinant rat PDE4A, PDE4B, and PDE4D proteins expressed in a heterologous system. These antibodies were used to characterize the cAMP-PDEs expressed in the rat brain. An immunoblot of extract of cortex and cerebellum demonstrated that at least seven different polypeptides specifically cross-reacted with the different antibodies, indicating that multiple cAMP-PDEs are expressed in this tissue. On the basis of cross-reactivity with PDE4D but not PDE4A or PDE4B antibodies, 93-and 105-kDa PDE4D species were detected in the cortex and cerebellum extract. These forms are different from the 68-kDa PDE4D form expressed in endocrine cells after hormonal stimulation. Although the 93-kDa form was recovered in both the soluble and particulate fractions, the 105-kDa polypeptide was mostly particulate in the cortex and cerebellum extracts. PDE4B forms of 90 -87 kDa were recovered in both soluble and particulate compartments of the brain extract. These forms were different from the previously identified PDE4A variants of 110 and 75 kDa. These data demonstrate that the presence of multiple cAMP-PDE genes is translated into cAMP-PDE proteins of different sizes and distinct immunological properties and that multiple variants derived from these cAMP-PDE genes are expressed in different regions of the brain and different subcellular compartments. These immunological tools will be useful to identify different cAMP-PDE forms expressed in organs targeted for pharmacological intervention with PDE4 inhibitors.The high affinity, cAMP-specific phosphodiesterases [type 4 according to the nomenclature proposed by Beavo et al. (1994)] are a class of enzymes with similar kinetic properties that are inhibited by the antidepressant rolipram and structurally related compounds. Although the presence of these forms has long been recognized, their distinctive properties are becoming evident only recently (Conti et al., 1995b). Early attempts to purify these forms have been hampered by their low abundance and instability (Conti and Swinnen, 1990). The molecular mass attributed to this group of enzymes ranges between 29 and 89 kDa (Conti and Swinnen, 1990). The definition of th...

The DISC locus in psychiatric illness

“…The PDE4s are abundantly expressed in the CNS and exhibit isoform-specific expression, [126][127][128][129][130] suggesting that each isoform has a specific role within the brain. Sites of expression include many regions implicated in the pathology of mental illness, such as the hippocampus, frontal cortex and nucleus accumbens.…”

Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice

“…Cortex and cerebellum were dissected from adult mouse brain and immediately homogenized as described (19). After centrifugation at 16,000 ϫ g for 20 min, the supernatant was immunoprecipitated with a PDE4D-specific mAb, M3S1, a PDE4A-specific polyclonal antibody, AC55, or a PDE4B-specific polyclonal antibody, K118 (19). The immunoprecipitated PDE4D, PDE4A, and PDE4B proteins were further detected by Western blot analysis using a PDE4D-specific mAb, F34-8F4, AC55, and K118, respectively.…”

Differential expression of cyclic nucleotide phosphodiesterases 4 in developing rat lung

“…Nonparametric Kruskal-Wallis multiple-group comparison and 2-group comparisons by Mann-Whitney U-test: *P < 0.05, significantly different from the F17 group; **P < 0.05, significantly different from the F1 group; y P < 0.05, significantly different from the F21 group. weight of 71 kDa, which may correspond to the PDE4B2 variant, the only variant currently described to present this migration pattern in the rat (Iona et al, 1998). Real-time PCR measurements of PDE4B2 mRNA steady-state demonstrated an expression pattern similar to that obtained with a generic pattern of PDE4B, implying that only PDE4B2 is expressed in the developing lung.…”

“…Membranes were blocked in 0.1% Tris Buffered Saline-Tween 20 containing 5% nonfat milk. Polyclonal rabbit anti-PDE4B antibody (1:2,500) (K118, kindly provided by Prof. Marco Iona et al, 1998), and monoclonal mouse anti-PDE4D antibody (1:10,000) (61D10E, ICOS Corp., Seattle WA) were used (Bolger et al, 1997). Second-step HRP-conjugated antibodies (1:7,000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and visualized by using ECL detection reagents (GE Healthcare Amersham).…”

“…They were then incubated in 1% PBS-BSA-10% donkey serum to block nonspecific binding. The primary antibody, a mouse monoclonal anti-PDE4D antibody (M3S1, provided by Prof. Marco Iona et al, 1998) diluted to 1:65 in PBS was added and incubated overnight at 4 C. The secondary antibody, an Alexa 488-conjugated donkey anti-mouse antibody (Molecular Probes, Invitrogen), diluted to 1:200, was added for 1 hr. Sections were also incubated with an anti-pro-SP-C primary antibody (C-19, Santa Cruz Biotechnology), diluted to 1:200, then a Rhodamine-conjugated donkey antigoat secondary antibody (TRITC: Jackson ImmunoResearch, Suffolk, UK), diluted to 1:200.…”