Characterization of a hormone-inducible, high affinity adenosine 3',5'-cyclic monophosphate phosphodiesterase from the rat Sertoli cell

Abstract

In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100,000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps. The major purified form of cAMP-PDE had a specific activity of 1-2 mumol/(min.mg of protein). Polyacrylamide gel electrophoresis and silver staining analysis showed that a 67-68 kDa polypeptide comigrated with the major peak of cAMP hydrolytic activity. The molecular weight of the crude or purified enzyme determined by gel filtration and sucrose density gradients was 150,000, suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a Km of 1.97 +/- 0.26 microM. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 microM. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The activity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxanthine > cilostamide = milrinone. Because mRNAs derived from two distinct PDE4B and PDE4D genes are present in the Sertoli cell, selective and nonselective PDE antibodies were used to determine the origin of the inducible PDE protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…This is believed to serve as negative-feedback regulation. Moreover, upregulation of certain PDE4 isoenzymes by chronic cAMP elevation occurs in some cells (72,94,281,367). FSH treatment of Sertoli cells causes cAMP-induced increases in PDE4D protein and mRNA levels, resulting in desensitization to subsequent FSH treatment.…”

Identification of cyclic AMP‐phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells

“…Western blot analyses were performed by using as primary antibody either the anti-PDE4 antiserum Kl16 or the anti-PDE4D monoclonal antibody M3S1 as previously described [29]. M3S1 was raised against a fusion protein obtained by linking the glutathione transferase to the carboxy terminal region of rat PDE4D and specifically recognizes the PDE4D isoforms, as detailed in [36]. The immunoreactive bands were detected by an enhanced chemiluminescence method, following the manufacturer's protocol (Amersham).…”

Expression of mRNA encoding cAMP‐specific phosphodiesterase isoforms in rat parotid glands

“…Follicle-stimulating hormone has enhanced inrracellular cAMP to induce the expression of PDE4 in Sertli cells (28). In this study, four of the PDE4 isozymes and also three alternative splicing variants of PDE4D, PDE4D1, PDE4D2 and PDE4D3 were detected in the parotid gland.…”

“…In the parotid gland, PDE4D1 was a major variant, and PDE4D2 and PDE4D3 were minor ones. Two mechanisms have been proposed for cAMP regulation by PDE, short-term and long-term regulations (6,22,23,27,28). In short-term regulation, the elevation of the intraccllular cAMP level may induce PDE activation through phosphorylation by A-kinase to resulting in the attenuation of the cAMP level.…”