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Cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase is functional in bovine mammary gland

V. Dostaler-Touchette^*, F. Bédard^*, C. Guillemette^*, F. Pothier^*, P. Y. Chouinard and F. J. Richard^*,1

^* Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Québec, Canada G1V 0A6
Département des Sciences Animales, Université Laval, Québec, Québec, Canada G1V 0A6

¹ Corresponding author: Francois.Richard{at}fsaa.ulaval.ca

Previous studies have shown that using nonspecific phosphodiesterase (PDE) inhibitors such as caffeine improved milk production, supporting the premise that modulation of intracellular concentration of cyclic nucleotides (cyclic AMP, cyclic guanosine 3'-5'-monophosphate) is involved. Intracellular cyclic nucleotides are degraded by the PDE enzyme family. The contribution of type IV PDE (PDE4) in the secretion of casein has been reported in rat mammary gland. The objective of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland. To understand the enzymatic expression pattern in the mammary gland, tissue samples were taken randomly from udders obtained from a local slaughterhouse. Reverse transcription PCR revealed that the PDE4D transcript was amplified, and the expected size fragment was obtained in a 1% agarose gel. Sequence analysis of the amplicon resulted in 99% homology to PDE4D. Moreover, Western blotting using a specific PDE4D antibody has confirmed that the protein of the isoenzyme PDE4D1 is present. A clear immunoreactive signal was also observed within the acini where epithelial cells are located. Assaying cyclic AMP PDE activity reported a total activity of 38.71 ± 3.22 fmol/min per µg of total protein. Rolipram, a specific PDE4 inhibitor, showed a sensitive activity of 8.48 ± 1.28 fmol/min per µg of total protein, indicating that PDE4 is responsible for one-fifth of the total enzymatic activity of PDE in the mammary gland. To further validate the presence of PDE4D in the bovine mammary epithelial cells, protein extracts from bovine mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected. The PDE assays reported a total activity of 30.16 ± 4.82 fmol/min per µg of total protein. Rolipram showed a sensible activity of 11.91 ± 5.93 fmol/min per µg of total protein. In conclusion, these results not only demonstrate the presence of PDE4D transcript and protein, but also show an active enzyme, suggesting a functional role of PDE4D in bovine mammary gland.

Key Words: dairy cow • phosphodiesterase • mammary gland