J. Dairy Sci. 2009. 92:3757-3765. doi:10.3168/jds.2009-2065
© 2009 American Dairy Science Association ®
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
 |
ABSTRACT
|
|---|
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
|
INTRODUCTION
|
|---|
One way to improve lactation would be to modulate the cyclic nucleotide [cyclic AMP (cAMP), cyclic guanosine 3'-5'-monophosphate (cGMP)] pathways. These key intracellular second messengers in many aspects of cellular function are hydrolyzed into their inactive form by the phosphodiesterase (PDE) enzyme family. This superfamily contains 11 families (PDE1 to PDE11) identified based on their substrate affinity, biochemical properties, regulation, and sensitivities to inhibitors (Beavo, 1995). Each family includes 1 to 4 distinct genes, which gives more than 20 genes encoding more than 80 different isoenzymes. Three PDE families are capable of hydrolyzing cAMP exclusively, 3 are cGMP-specific, and the remaining 5 degrade both cyclic nucleotides.
Type IV PDE family (PDE4) is cAMP-specific and is inhibited by rolipram (Lugnier et al., 1983, 1986). It is currently the largest PDE family with 4 genes (PDE4A, PDE4B, PDE4C, and PDE4D), most of which contain 2 or more alternative splice variants characterized by unique N-terminal regions (Beavo, 1995; Houslay et al., 2005). Although the correlation between the level of PDE4 and cAMP concentration is unclear, this family is considered an important cAMP homeostatic regulator (Louis and Baldwin, 1974; Conti et al., 2003). The PDE4 family is involved in various functional activities such as cell desensitization or adaptation, signaling cross-talk, and cAMP signal compartmentalization (Conti et al., 2003). Furthermore, this regulation can be on a short- or long-term basis depending on the isoenzyme involved. The long form PDE4 contains a protein kinase A–mediated phosphorylation site in their targeting domain and will be activated more quickly than the short forms. The long-term regulation occurs at the transcriptional level rather than by phosphorylation (Conti et al., 2003).
The PDE4 family is widely expressed in various tissues, but particular attention has been oriented toward the brain, inflammatory cells, cardiovascular tissues, smooth muscle, and testis (Richter et al., 2005; Li et al., 2006; Braun et al., 2007; Schmitz et al., 2007; Hirose et al., 2008). Recently, 3 type IV isoenzymes, RNPDE4A5, RNPDE4A8, and RNPDE4D3, have been identified in the rat mammary gland (Pooley, 2002). Moreover, early studies have shown that using nonspecific PDE inhibitors such as caffeine improved milk production (Sheffield, 1991). The effects of rolipram, a highly PDE4-specific inhibitor, have been studied in multiple experimental designs involving different cell types. Nevertheless, its efficiency on the bovine mammary gland or on milk production has never been reported in the literature.
We hypothesized that the enzymatic activity of certain families of phosphodiesterases plays a critical role in dairy cow lactation. The aim of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland.
|
MATERIALS AND METHODS
|
|---|
Tissue Collection
The rear quarters of 4 bovine mammary glands obtained from a local slaughterhouse were used in a series of experimental procedures. A first portion of the mammary tissue collected was immediately flash-frozen in liquid N2 after being harvested and was used for reverse transcription (RT)-PCR studies.
For mammary gland epithelial cell isolation procedures, a second portion of the glandular area of the udder was washed 2 times in 70% ethanol and immediately brought to our laboratories in Eagle Spinner Salts Solution (ESS; Sigma-Aldrich, St. Louis, MO) plus antibiotics (100 µg/mL streptomycin and 100 µg/mL gentamicin, Sigma-Aldrich) on ice. The inner part of the piece of tissue was used for enzymatic digestion.
A third portion of bovine mammary gland tissue was transported on ice and immediately homogenized in hypotonic buffer [20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2 mM ethylene glycol tetraacetic acid, 50 mM sodium fluoride, 50 mM benzamidine, 10 mM sodium pyrophosphate, 4 µg/mL aprotinin, 0.7 µg/mL pepstatin, 10 µg/mL soybean trypsin inhibitor, 0.5 µg/mL leupeptin, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% Triton X-100]. Tris-HCl was purchased from Fisher Scientific Ltd. (Nepean, Ontario, Canada) and all other products from Sigma-Aldrich. The homogenate was centrifuged for 20 min at 13,000 x g to obtain the supernatant, which was used for PDE assay and Western blotting.
RNA Extraction and RT-PCR
Extraction of RNA was carried out using a Trizol Reagent (Invitrogen Canada Inc., Burlington, Ontario, Canada) followed by an Absolutely RNA Microprep Kit from Stratagene (La Jolla, CA) according to the manufacturers’ protocols. The RNA samples were eluted in 15 µL followed by reverse transcription using an OmniScript RT Kit from Qiagen (Mississauga, Ontario, Canada) and Oligo d(T) primer from Ambion (Austin, TX). To each tube, a mixture containing 2 µL of Omniscript Buffer (5x; Qiagen), 2 µL of dNTPs (50 µM; Qiagen), 1 µL of SUPERase-In (20 U/µL; Ambion), 1 µL of Oligo d(T) Primer RETROscript (Ambion), and 1 µL of Omniscript Reverse Transcriptase (Qiagen) was added. The mixture was then incubated at 42°C for 2 h. The primer pairs (Integrated DNA Technologies, Skokie, IL) were designed based on 
-casein (BC_102120.1), human PDE4A (BC_038234.1), human PDE4B (NM_002600.2), human PDE4C (NM_000923.1), and human PDE4D (NM_006203.2) sequences as described in Table 1.
The PCR reactions were carried out in a 50-µL reaction volume using Taq polymerase from New England Biolabs (Ipswich, MA). The following cycling conditions were used for all amplifications: 2 min at 95°C [1 min at 95°C, 1 min at 60°C, 1 min at 72°C] 40 times, and 10 min at 72°C. Additional amplifications were performed on an equivalent amount of RNA to exclude genomic DNA contamination. The PCR products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining.
The PCR products producing amplicons of the expected size were purified using a MinElute PCR Purification Kit (Qiagen) according to the manufacturer’s protocol and sequenced with a 16-capillary genetic analyzer (biomolecular analysis platform, Université Laval, Québec, Canada). The obtained sequences were compared with corresponding PDE mRNA.
Western Blotting
Homogenized tissues were loaded in sample buffer [60 mM Tris-HCl (pH 6.8; Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada), 10.5% (vol/vol) glycerol (Sigma-Aldrich), 2% (wt/vol) SDS (Laboratoire Mat Inc., Beauport, Québec, Canada), 0.005% (wt/vol) bromophenol blue (Sigma-Aldrich), and 5% (vol/vol) 2-mercaptoethanol (Sigma-Aldrich)] onto a 12% polyacrylamide gel for electrophoresis. The samples were then transferred onto a Hybond-P membrane (GE Healthcare, Baie d’Urfé, Québec, Canada) using a Mini Protean 3 Cell apparatus (Bio-Rad Laboratories Ltd.). Membranes were blocked for 60 min with Tris-buffered saline [150 mM NaCl, 10 mM Tris-HCl (pH 7.4)] containing 0.1% (vol/vol) Tween 20 (TTBS) and 5% (vol/vol) blocking reagent (skim milk). The first hybridization was performed during 2 h at room temperature in the TTBS containing the primary antibody, anti-PDE4D (no. sc-25100; Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted at 1:200. The membranes were then washed 3 times in TTBS and hybridized with the secondary antibody, rabbit anti-goat IgG peroxidase-conjugated (Fabgennix International Inc., Frisco, TX) diluted at 1:30,000 in TTBS for 45 min at room temperature. Proteins were detected using an ECL Western Blotting Detection Reagent (GE Healthcare) and exposed on autoradiographic films (GE Healthcare). Images were analyzed using Quantity One Software (Bio-Rad Laboratories Ltd.). A stripping solution (25 mM glycine-HCl, pH 2; BDH Industries Ltd., Mumbai, India; 1% SDS; Laboratoire Mat Inc.) was used to remove the hybridized antibody to allow subsequent hybridization. Antibody preincubation experiments were performed with a peptide mapping near the C-terminus of human PDE4D (no. sc-25100P; Santa Cruz Biotechnology Inc.).
Immunohistochemistry
Mammary gland explants were fixed in Bouin fixation solution and mounted in paraffin blocks. Immunohistochemistry was performed on 10-µm-thick sections. The slides were immersed in baths containing 1% lithium carbonate in 70% ethanol to inactivate the residual picric acid from Bouin solution, and free aldehydes were blocked using 300 mM glycine (pH 7.4; Pelletier et al., 1999). The mammary gland sections were washed in PBS (1.5 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), and endogenous peroxidase activity was inhibited by 3% H2O2 in methanol for 10 min. Antigen retrieval was achieved by incubation of the slides for 10 min in a boiling bath containing 10 mM sodium citrate pH 6.0. Nonspecific sites were blocked with 1% BSA in PBS for 1 h. The slides were then incubated for 2 h at room temperature in the presence of the primary antibody, anti-PDE4D (no. PD4-401AP; Fabgennix International Inc.) diluted at 1:150 in the blocking solution. After being washed 5 times with PBS supplemented with 0.05% Tween-20, the sections were incubated with a biotin-conjugated goat anti-rabbit IgG (1:1,000) in the blocking solution for 1 h at room temperature, washed again in PBS supplemented with 0.05% Tween-20, and incubated with peroxidase-conjugated streptavidin for 30 min at room temperature (both from Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The slides were extensively washed in PBS supplemented with 0.05% Tween-20 and the immune complex was revealed with 3,3'-diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin. At last, the tissue sections were mounted in Permount (Fisher Scientific Ltd.) and observed by light microscopy. Mammary gland sections incubated with the same concentration of nonimmune rabbit IgG (Jackson ImmunoResearch Laboratories Inc.) were used for nonspecific labeling.
PDE Assay
Phosphodiesterase activity was measured in 11 replicates from 4 different bovine mammary glands and in epithelial cells isolated from 2 different bovine mammary glands. The assay was conducted at 34°C in a 200-µL final volume with 1 µM cAMP as substrate, following the method of Thompson et al. (1979). The solution composition consisted of 40 mM Tris-HCl (pH 8.0; Fisher Scientific Ltd.), 10 mM MgCl2 (Sigma-Aldrich), 5 mM 2-mercaptoethanol (Sigma-Aldrich), 0.75 mg/mL BSA (fraction V; Sigma-Aldrich), 1 µM cold cyclic AMP (Sigma-Aldrich), and [3H] cyclic AMP (1 x 105 cpm/tube; 30 Ci/mmol; GE Healthcare). The measurements were performed in the presence of PDE inhibitors 3-isobutyl-methylxanthine (IBMX; 1 mM, nonspecific; Sigma-Aldrich), cilostamide (10 µM, PDE3-specific; Biomol, Plymouth Meeting, PA), rolipram (10 µM, PDE4-specific; Biomol), dipyridamole (30 µM, nonspecific; Sigma-Aldrich), and papaverine (400 nM, PDE10-specific; Sigma-Aldrich). The Ca2+/calmodulin-stimulated PDE1 activity was measured using EGTA (2 mM; Sigma-Aldrich), calmodulin (4 µg/mL; Sigma-Aldrich), and trifluoperazine (12.5 µM; Sigma-Aldrich). The IBMX-, cilostamide-, rolipram-, dipyridamole-, and papaverine-sensitive PDE activities were obtained by subtracting the PDE activity measured in the presence of the respective inhibitors from the total activity. Data are presented as mean ± SD.
Cell Isolation
Mammary gland epithelial cells were isolated as described previously (Morin et al., 2005), with slight modifications. Samples of tissue of approximately 10 g were washed twice in 70% ethanol under sterile conditions. Tissue slices (1–2 mm thick) were then cut inside the tissue sample to eliminate the surfaces in contact with nonsterile atmosphere. These slices were repeatedly washed with ESS plus antibiotics (as described above) and 1% fetal calf serum (FCS; Hyclone, Logan, UT) at room temperature until the washing solution remained clear and then minced between scalpels into 1-mm3 fragments.
Dissociation medium composed of ESS plus antibiotics, 5% FCS, and pronase E (5.5 U/mL, Streptomyces griseus; Sigma-Aldrich) was added (4 mL/g of wet tissue). Incubation with enzyme occurred at 37°C for 1 h. Every 10 min, the mixture was passed through a 25-mL pipette. After incubation, 10 mL of ESS plus antibiotics and 3% FCS was added to stop the reaction.
The suspension was filtered through a sterile stainless steel mesh, washed twice by centrifugation (5 min at 3,000 x g), and suspended again in 1 mL of ESS plus antibiotics and 3% FCS to remove enzymes and contaminants. These steps were repeated 3 times. Cells were then placed in a 15-mL Falcon tube (Becton Dickinson Biosciences, Mississauga, Ontario, Canada) and incubated for 30 min at 37°C. Viability estimated by trypan-blue exclusion was more than 85%. Cells were flash-frozen in liquid N2 and stored at –80°C until analysis.
|
RESULTS
|
|---|
The type IV PDE family is encoded by 4 separate genes: 4A, 4B, 4C, and 4D. To assess which members of the type IV family are expressed in the bovine mammary gland, RT-PCR studies were undertaken. All of the 4 genes known to be expressed in this family were detected with one clear PCR product visualized on an agarose gel (Figure 1). To confirm the identity of the amplification products, they were sequenced and found to have 97% homology to bovine, 87% homology to human, 99% homology to bovine, and 99% homology to bovine sequences for PDE4A, 4B, 4C, and 4D, respectively (Table 2) supporting the premise that the 4 genes of PDE4 are expressed in bovine mammary gland.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 1. Detection of type IV phosphodiesterase (PDE4) family gene products by reverse transcription (RT)-PCR. Reaction products were resolved by electrophoresis on a 1% agarose gel and visualized with ethidium bromide under UV light. The RNA was prepared from bovine mammary tissue and probed for the expression of the 4 known gene families, 4A, 4B, 4C, and 4D of the type IV cyclic AMP phosphodiesterase by RT-PCR as described in Materials and Methods. Lane –RT = RNA; – = lanes without cDNA; c = lanes with cDNA; K-C = -casein; 4A = PDE4A; 4B = PDE4B; 4C = PDE4C; 4D = PDE4D.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Sequence homology percentage of amplified fragments of expected size with the corresponding phosphodiesterase (PDE) gene transcripts
|
|
Protein extracts from bovine mammary gland were separated on SDS-PAGE gels, and PDE4D protein was detected (Figure 2A). In mammary gland, the immunoreactive band was detected at a molecular weight around 75 kDa. A band was also detected at the same molecular weight in testis protein extract. Densitometric analysis of this band compared with 
-tubulin revealed a 1:4 ratio (Figure 2C and 2D). To confirm the identity of the 75-kDa immunoreactive band as PDE4D, the membrane was stripped and a second hybridization was conducted. Identical conditions were employed, except that the anti-PDE4D antibody was preincubated with the PDE4D peptide used to raise the antibody. This second hybridization displayed no immunoreactive band (Figure 2B). To demonstrate that the absence of immunoreactive band was not due to the stripping method, another membrane with the same samples was hybridized for the first time with the PDE4D antibody preincubated with the PDE4D peptide. No 75-kDa immunoreactive band was detected (data not shown).
Because mammary gland is composed of several types of cells, the expression of PDE4D was assessed by immunohistochemistry on bovine mammary gland. The enzyme is expressed in epithelial cells surrounding the acini (Figure 3). A clear immunoreactive signal was observed within the acini where epithelial cells are located, supporting the expression of PDE4D.
View larger version (78K):
[in this window]
[in a new window]
|
Figure 3. Expression of type IV phosphodiesterase (PDE4D) in bovine mammary gland. Bovine mammary gland was fixed in Bouin’s solution and embedded in paraffin. Sections of 10 µm were used for immunohistochemistry using the PDE4D polyclonal antibody. Immunodetection was performed using a biotinylated secondary antibody and peroxidase-conjugated streptavidin. The cellular localization of PDE4D was visualized with 3,3'-Diaminobenzidine. A) No signal was observed when rabbit IgG were used (200x). B) PDE4D expression at 200x. The arrow is showing expression in epithelial cells.
|
|
To determine whether PDE4 is functional in mammary gland, PDE activity was assayed using specific PDE inhibitor. Rolipram (PDE4-specific) was responsible for 20% of the total cAMP-PDE activity measured in mammary gland tissue. Cilostamide (PDE3-specific) and papavarine (PDE10-specific) were both responsible for approximately one-fifth of the total PDE activity. Total cAMP-PDE activity was inhibited by 91% using IBMX, suggesting low (<10%) PDE8 activity, which is insensitive to this inhibitor (Table 3). No increase in the total activity of the homogenates was detected when calmodulin was included in the assay, indicating that PDE1 activity was below the detectable range. These data support the functional presence of the PDE4 enzyme.
View this table:
[in this window]
[in a new window]
|
Table 3. Effect of selective phosphodiesterase (PDE) inhibitors and activators on PDE activity of bovine mammary gland explants1
|
|
To further validate the presence of PDE4D in bovine mammary epithelial cells, protein extracts from bovine testis, mammary gland, and mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected (Figure 4A). In mammary epithelial cells, an immunoreactive band was detected at a molecular weight similar to that in testis protein extract and mammary gland. Densitometric analysis of the 75-kDa PDE4D band compared with -tubulin revealed a 1:3 ratio of PDE4D over tubulin (Figure 4B and 4C).
Cyclic AMP-PDE activity was measured in bovine mammary epithelial cells isolated from 2 different mammary gland (Table 4). Rolipram (PDE4-specific) was responsible for 40% of the total cAMP-PDE activity measured in mammary epithelial cells. Cilostamide (PDE3-specific) was responsible for 20% of total PDE activity. Total activity was inhibited at 95% by the nonspecific inhibiter IBMX, suggesting low PDE8 activity, which is IBMX-insensitive. Papaverine-sensitive activity was almost absent. These data support the premise that the PDE4 family is the most active PDE in mammary epithelial cells.
View this table:
[in this window]
[in a new window]
|
Table 4. Effect of selective phosphodiesterase (PDE) inhibitors and activators on PDE activity of bovine mammary epithelial cells1
|
|
|
DISCUSSION
|
|---|
This study clearly demonstrates for the first time the functional presence of type IV cyclic nucleotide PDE family in bovine mammary gland tissue as well as in epithelial cells. Several approaches were used including enzymatic assay, Western blotting, and RT-PCR.
As early as 1975, several reports suggested the efficacy of cAMP-elevating agents in stimulating the rate of casein secretion in ewe, rabbit, and bovine mammary glands (Ollivier-Bousquet and Denamur, 1975; Park et al., 1979). A decade later, Brown and Harris (1988) reported the effects of intravenously infused caffeine (a nonspecific PDE inhibitor) on reversing the milk fat reduction in dairy goats. A few years later, caffeine was given to mice during pregnancy and the cell number of mammary gland was increased (Sheffield, 1991) suggesting a mammogenic effect of caffeine and an enhanced lactational performance. More recently, a specific role for PDE4 activity (cAMP-PDE) in the regulation of casein secretion was suggested in rat mammary gland (Pooley, 2002). Pooley (2002) showed that more than 70% of PDE activity measured was type IV specific. Hence, PDE4 was a good candidate to be investigated in the bovine mammary gland.
As for its presence, RT-PCR studies amplified the 4 known transcripts (A, B, C, and D). This is the first study reporting family-specific PDE expression in bovine mammary gland. These data are consistent with the results presented by Pooley (2002) on lactating rat mammary gland. In the rat, PDE4A and PDE4D are the only subfamilies reported. A difference is therefore notable because the PDE4B and PDE4C transcripts were also obtained in the bovine mammary gland. Variants PDE4A5 and PDE4A8 were identified by Pooley (2002) in the rat mammary gland. Interestingly, the long form PDE4A5 has a role in the prevention of cell apoptosis (Houslay and Adams, 2003). Because the apoptosis of the epithelial cells in mammary gland occurs during the dry period, PDE4A5 may be involved during lactation, at least in the last months. It has also been demonstrated that short forms of PDE4 such as PDE4B2, PDE4D1, and PDE4D2 play a role in myometrial cells (Mehats et al., 1999, 2001). In particular, the short form PDE4B2 is implicated in an increased cAMP degradation in near-term pregnancy myometrium (Mehats et al., 2001). These short forms are mainly transcriptionally regulated (Houslay et al., 2007).
The presence of 2 domains N-terminal to the catalytic domain called upstream conserved region 1 and 2 (UCR1/2) is a structural feature unique to the cAMP-specific PDE4 (Richter and Conti, 2002). The presence or not of UCR1 determines the length of the variants (Conti et al., 2003; Richter et al., 2005). Long forms, which are protein kinase A-mediated, are phosphorylated at a site present on the UCR1 domain and are identified as short-term regulation PDE (Richter et al., 2005). One the other hand, the short forms, which can present a complete (short forms) or partial (super-short forms) UCR2, are regulated at the transcriptional level and are responsible for regulation on a long-term basis (Beavo, 1995). Immunodetection confirmed the presence of PDE4D protein in the mammary gland (Figure 2). Richter et al. (2005) demonstrated that the splice variants PDE4D1, PDE4D2, and PDE4D3 have a theoretical molecular mass of 66.2, 57.6, and 76.3 but migrate at 78.5 ± 1.3, 68.2 ± 2.0, and 89.7 ± 1.8 kDa, respectively. The isoform observed in the present study has the same migration behavior on SDS-PAGE as PDE4D1 detected in rat COS7 cells (Richter et al., 2005) and in mouse NIH-3T3 cells (Chandrasekaran et al., 2008). From the study of one of the PDE4 expressed in the mammary gland, cAMP intracellular content of the mammary gland cell appears to be long-term regulated via PDE4D1. This result is not surprising because milk production occurs over a long period calculated in months.
The type IV PDE is not only present but active because it is responsible for approximately 20% of the total PDE activity in the gland and 40% in mammary epithelial cells (Tables 3 and 4). The observed difference between the whole tissue (Table 3) and the isolated epithelial cells (Table 4) is mainly explained by the presence of nonepithelial cells in the whole tissue samples. This is illustrated by PDE activity measured in stromal and fat cells (data not shown). Because the analyzed samples are composed of different cell types, immunolocalization of PDE4D in epithelial cells would support a functional role. Because milk proteins are secreted in the acini lumen by epithelial cells, the immunodetection is supportive of a functional significance (Linzell and Peaker, 1971; Franke et al., 1976).
Generally speaking, cows’ milk is composed of 87.6% water, 4.6% lactose, 3.7% fat, 3.4% proteins (2.8% casein), and 0.7% minerals (Jensen, 1995). Of the proteins, about 80% are caseins. Casein secretion has been observed to be specifically regulated by PDE4 in rat mammary gland (Pooley, 2002) supporting the idea that PDE4 is an important functional enzyme of the bovine mammary gland.
Other PDE appear to be functional in the bovine mammary gland such as PDE3 and PDE10. The cilostamide-sensitive PDE (PDE3) might be implicated in milk fat production because it is stimulated by insulin in adipocytes (Enoksson et al., 1998). This hormone stimulates a cAMP phosphodiesterase in acini isolated from mammary tissue of lactating rat (Aitchison et al., 1984). The type 10 PDE family was discovered in 1999 and is known to be highly expressed in brain and testis of various species (Strick et al., 2006). We propose that this PDE family (Strick et al., 2006) may have a role in the bovine mammary gland, but this needs to be further investigated. Finally, a low IBMX-insensitive PDE activity is observed in the tissue as well as in the epithelial cells suggesting the functional presence of PDE8. Further investigations are needed to assess the physiological relevance of these family-specific PDE in lactopoiesis.
In conclusion, this study described for the first time the functional presence of the PDE4 family in the bovine mammary gland. Establishment of some PDE profiles, including PDE4D, during a complete lactation needs to be done to understand their physiological roles in mammary tissue in relation to the synthesis and secretion of individual milk constituents.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by the Dairy Farmers of Canada and by the Natural Sciences and Engineering Research Council of Canada. We thank Richard Prince (Université Laval) for harvesting the udders at the slaughterhouse. The assistance of the following students and colleagues at Université Laval in implementing the study is also gratefully acknowledged and appreciated: Maxime Sasseville, Marie-Claude Gagnon, Guillaume Morin, and Josée Mercier.
Received for publication January 22, 2009.
Accepted for publication April 16, 2009.
|
REFERENCES
|
|---|
Aitchison, R., West, D. W. and Clegg, R. A.. 1984. Insulin-stimulated high affinity cyclic AMP phosphodiesterase in rat mammary acini. FEBS Lett. 167:25–28.[CrossRef][Medline]
Beavo, J. A. 1995. Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms. Physiol. Rev. 75:725–748.[Abstract/Free Full Text]
Braun, N. N., Reutiman, T. J., Lee, S., Folsom, T. D. and Fatemi, S. H.. 2007. Expression of phosphodiesterase 4 is altered in the brains of subjects with autism. Neuroreport 18:1841–1844.[CrossRef][Medline]
Brown, D. L. and Harris, D. M.. 1988. Effects of caffeine and 3-isobutyl 1-methyl xanthine on caprine milk secretion. J. Dairy Sci. 71:513–517.[Abstract/Free Full Text]
Chandrasekaran, A., Toh, K. Y., Low, S. H., Tay, S. K. H., Brenner, S. and Goh, D. L. M.. 2008. Identification and characterization of novel mouse PDE4D isoforms: Molecular cloning, subcellular distribution and detection of isoform-specific intracellular localization signals. Cell. Signal. 20:139–153.[CrossRef][Medline]
Conti, M., Richter, W., Mehats, C., Livera, G., Park, J. Y. and Jin, C.. 2003. Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J. Biol. Chem. 278:5493–5496.[Free Full Text]
Enoksson, S., Degerman, E., Hagström-Toft, E., Large, V. and Arner, P.. 1998. Various phosphodiesterase subtypes mediate the in vivo antilipolytic effect of insulin on adipose tissue and skeletal muscle in man. Diabetologia 41:560–568.[CrossRef][Medline]
Franke, W. W., Luder, M. R., Kartenbeck, J., Zerban, H. and Keenan, T. W.. 1976. Involvement of vesicle coat material in casein secretion and surface regeneration. J. Cell Biol. 69:173–195.[Abstract/Free Full Text]
Hirose, R., Manabe, H., Yanagawa, K., Ohshima, E. and Ichimura, M.. 2008. Differential effects of PDE4 inhibitors on cortical neurons and T-lymphocytes. J. Pharmacol. Sci. 106:310–317.[CrossRef][Medline]
Houslay, M. D. and Adams, D. R.. 2003. PDE4 cAMP phosphodiesterases: Modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem. J. 370:1–18.[CrossRef][Medline]
Houslay, M. D., Baillie, G. S. and Maurice, D. H.. 2007. cAMP-specific phosphodiesterase-4 enzymes in the cardiovascular system: A molecular toolbox for generating compartmentalized cAMP signaling. Circ. Res. 100:950–966.[Abstract/Free Full Text]
Houslay, M. D., Schafer, P. and Zhang, K. Y. J.. 2005. Keynote review: Phosphodiesterase-4 as a therapeutic target. Drug Discov. Today 10:1503–1519.[CrossRef][Medline]
Jensen, R. G. 1995. Handbook of Milk Composition. Academic Press, San Diego, CA.
Li, X., Huston, E., Lynch, M. J., Houslay, M. D. and Baillie, G. S.. 2006. Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293Î22 cells and cardiac myocytes. Biochem. J. 394:427–435.[CrossRef][Medline]
Linzell, J. L. and Peaker, M.. 1971. Mechanism of milk secretion. Physiol. Rev. 51:564–597.[Free Full Text]
Louis, S. L. and Baldwin, R. L.. 1974. Effect of adrenalectomy and insulin insufficiency upon the adenosine 3', 5' cyclic monophosphate system of the rat mammary glands. J. Dairy Sci. 58:502–506.
Lugnier, C., Schoeffter, P., Le Bec, A., Strouthou, E. and Stoclet, J. C.. 1986. Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta. Biochem. Pharmacol. 35:1743–1751.[CrossRef][Medline]
Lugnier, C., Stierle, A., Beretz, A., Schoeffter, P., Lebec, A., Wermuth, C. G., Cazenave, J. P. and Stoclet, J. C.. 1983. Tissue and substrate specificity of inhibition by alkoxy-aryl-lactams of platelet and arterial smooth muscle cyclic nucleotide phosphodiesterases relationship to pharmacological activity. Biochem. Biophys. Res. Commun. 113:954–959.[CrossRef][Medline]
Mehats, C., Tanguy, G., Dallot, E., Cabrol, D., Ferre, F. and Leroy, M. J.. 2001. Is up-regulation of phosphodiesterase 4 activity by PGE2 involved in the desensitization of β-mimetics in late pregnancy human myometrium? J. Clin. Endocrinol. Metab. 86:5358–5365.[Abstract/Free Full Text]
Mehats, C., Tanguy, G., Dallot, E., Robert, B., Rebourcet, R., Ferre, F. and Leroy, M. J.. 1999. Selective up-regulation of phosphodiesterase-4 cyclic adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase variants by elevated cAMP content in human myometrial cells in culture. Endocrinology 140:3228–3237.[Abstract/Free Full Text]
Morin, G., Lalancette, C., Sullivan, R. and Leclerc, P.. 2005. Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit. Mol. Reprod. Dev. 71:523–534.[CrossRef][Medline]
Ollivier-Bousquet, M. and Denamur, R.. 1975. Effet de l'état physiologique et du 3'5' adenosine monophosphate cyclique sur le transit intracellulaire et l'excrétion des protéines du lait. Étude autoradiographique en microscopie électronique. J. Microsc. Biol. Cell. 23:62–68.
Park, C. S., Smith, J. J., Eigel, W. N. and Keenan, T. W.. 1979. Selected hormonal effects on protein secretion and amino acid uptake by acini from bovine mammary gland. Int. J. Biochem. 10:889–894.[CrossRef][Medline]
Pelletier, R. M., Trifaro, J. M., Carbajal, M. E., Okawara, Y. and Vitale, M. L.. 1999. Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa. Biol. Reprod. 60:1128–1136.[Abstract/Free Full Text]
Pooley, L. 2002. Type IV phosphodiesterase activity specifically regulates cAMP-stimulated casein secretion in the rat mammary gland. Biochim. Biophys. Acta 1590:84–92.[Medline]
Richter, W. and Conti, M.. 2002. Dimerization of the type 4 cAMP-specific phosphodiesterases ss mediated by the upstream conserved regions (UCRs). J. Biol. Chem. 277:40212–40221.[Abstract/Free Full Text]
Richter, W., Jin, S. L. and Conti, M.. 2005. Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue. Biochem. J. 388:803–811.[CrossRef][Medline]
Schmitz, T., Souil, E., Herve, R., Nicco, C., Batteux, F., Germain, G., Cabrol, D., Evain-Brion, D., Leroy, M. J. and Mehats, C.. 2007. PDE4 inhibition prevents preterm delivery induced by an intrauterine inflammation. J. Immunol. 178:1115–1121.[Abstract/Free Full Text]
Sheffield, L. G. 1991. Caffeine administered during pregnancy augments subsequent lactation in mice. J. Anim. Sci. 69:1128–1132.[Abstract]
Strick, C. A., C. J. Schmidt, and F. S. Menniti. 2006. PDE10A: A striatum-enriched, dual-substrate phosphodiesterase. Pages 237–254 in Cyclic Nucleotide Phosphodiesterases in Health and Disease. J. A. Beavo, S. H. Francis, and M. D. Houslay, ed. CRC Press, Boca Raton, FL.
Thompson, W. J., Terasaki, W. L., Epstein, P. M. and Strada, S. J.. 1979. Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme. Adv. Cyclic Nucleotide Res. 10:69–92.[Medline]
TOP
ABSTRACT
INTRODUCTION
