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• W1993171057 abstract "In this study, we describe a novel mechanism by which a protein kinase C (PKC)-mediated activation of the Raf-extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade regulates the activity and membrane targeting of members of the cyclic AMP-specific phosphodiesterase D family (PDE4D). Using a combination of pharmacological and biochemical approaches, we show that increases in intracellular cAMP cause a protein kinase A-mediated phosphorylation and activation of the two PDE4D variants expressed in vascular smooth muscle cells, namely PDE4D3 and PDE4D5. In addition, we show that stimulation of PKC via the associated activation of the Raf-MEK-ERK cascade results in the phosphorylation and activation of PDE4D3 in these cells. Furthermore, our studies demonstrate that simultaneous activation of both the protein kinase A and PKC-Raf-MEK-ERK pathways allows for a coordinated activation of PDE4D3 and for the translocation of the particulate PDE4D3 to the cytosolic fraction of these cells. These data are presented and discussed in the context of the activation of the Raf-MEK-ERK cascade acting to modulate the activation and subcellular targeting of PDE4D gene products mediated by cAMP. In this study, we describe a novel mechanism by which a protein kinase C (PKC)-mediated activation of the Raf-extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade regulates the activity and membrane targeting of members of the cyclic AMP-specific phosphodiesterase D family (PDE4D). Using a combination of pharmacological and biochemical approaches, we show that increases in intracellular cAMP cause a protein kinase A-mediated phosphorylation and activation of the two PDE4D variants expressed in vascular smooth muscle cells, namely PDE4D3 and PDE4D5. In addition, we show that stimulation of PKC via the associated activation of the Raf-MEK-ERK cascade results in the phosphorylation and activation of PDE4D3 in these cells. Furthermore, our studies demonstrate that simultaneous activation of both the protein kinase A and PKC-Raf-MEK-ERK pathways allows for a coordinated activation of PDE4D3 and for the translocation of the particulate PDE4D3 to the cytosolic fraction of these cells. These data are presented and discussed in the context of the activation of the Raf-MEK-ERK cascade acting to modulate the activation and subcellular targeting of PDE4D gene products mediated by cAMP. phosphodiesterase angiotensin II calf intestinal alkaline phosphatase extracellular signal-regulated kinase Hanks' balanced salt solution mitogen-activated protein kinase ERK kinase polyacrylamide gel electrophoresis protein kinase A protein kinase C phorbol 12-myristate 13-acetate vascular smooth muscle cell Cyclic nucleotide phosphodiesterases (PDEs)1 catalyze the hydrolysis of adenosine cyclic 3′,5′-monophosphate (cAMP) and of guanosine cyclic 3′,5′-monophosphate (cGMP) to their respective 5′ nucleoside monophosphates and play a central role in regulating cyclic nucleotide-mediated cell signaling (1Beavo J.A. Reifsnyder D.H. Trends Pharmacol. Sci. 1990; 11: 150-155Abstract Full Text PDF PubMed Scopus (827) Google Scholar, 2Bolger G. Michaeli T. Martins T. St. John T. Steiner B. Rodgers L. Riggs M. Wigler M. Ferguson K. Mol. Cell. Biol. 1993; 13: 6558-6571Crossref PubMed Scopus (286) Google Scholar, 3Manganiello V.C. Taira M. Degerman E. Belfrage P. Cell Signal. 1995; 7: 445-455Crossref PubMed Scopus (122) Google Scholar, 4Loughney K. Ferguson K. Schudt C. Dent G. Rabe K.F. The Handbook of Immunopharmacology: Phosphodiesterase Inhibitors. Academic Press, London1996: 1-14Google Scholar, 5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar). PDEs form a multigene family, with individual members classified using several criteria including substrate selectivity, inhibitor sensitivity, and molecular sequence (1Beavo J.A. Reifsnyder D.H. Trends Pharmacol. Sci. 1990; 11: 150-155Abstract Full Text PDF PubMed Scopus (827) Google Scholar, 2Bolger G. Michaeli T. Martins T. St. John T. Steiner B. Rodgers L. Riggs M. Wigler M. Ferguson K. Mol. Cell. Biol. 1993; 13: 6558-6571Crossref PubMed Scopus (286) Google Scholar, 3Manganiello V.C. Taira M. Degerman E. Belfrage P. Cell Signal. 1995; 7: 445-455Crossref PubMed Scopus (122) Google Scholar, 4Loughney K. Ferguson K. Schudt C. Dent G. Rabe K.F. The Handbook of Immunopharmacology: Phosphodiesterase Inhibitors. Academic Press, London1996: 1-14Google Scholar, 5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar). Individual members of each individual PDE type are encoded by as many as four different genes, each of which can in turn give rise to variants by alternate splicing of mRNA or the use of alternate promoters (2Bolger G. Michaeli T. Martins T. St. John T. Steiner B. Rodgers L. Riggs M. Wigler M. Ferguson K. Mol. Cell. Biol. 1993; 13: 6558-6571Crossref PubMed Scopus (286) Google Scholar, 3Manganiello V.C. Taira M. Degerman E. Belfrage P. Cell Signal. 1995; 7: 445-455Crossref PubMed Scopus (122) Google Scholar, 4Loughney K. Ferguson K. Schudt C. Dent G. Rabe K.F. The Handbook of Immunopharmacology: Phosphodiesterase Inhibitors. Academic Press, London1996: 1-14Google Scholar, 5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar). Recently, significant progress has been made in elucidating some of the mechanisms regulating the activity and expression of some of the PDE. In this regard, one family that has received a significant amount of attention has been the cAMP-specific, Rolipram-inhibited PDE4 (2Bolger G. Michaeli T. Martins T. St. John T. Steiner B. Rodgers L. Riggs M. Wigler M. Ferguson K. Mol. Cell. Biol. 1993; 13: 6558-6571Crossref PubMed Scopus (286) Google Scholar, 4Loughney K. Ferguson K. Schudt C. Dent G. Rabe K.F. The Handbook of Immunopharmacology: Phosphodiesterase Inhibitors. Academic Press, London1996: 1-14Google Scholar,5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar). In human, rat and mouse, four distinct genes encode PDE4 (PDE4A, PDE4B, PDE4C, andPDE4D), with each, as a result of alternate splicing or the use of alternate promoters, potentially giving rise to multiple enzyme variants. As a result of early work by several laboratories, it has been established that several PDE4 variants can be expressed in individual tissues and that these genes are regulated by transcriptional and/or posttranslational mechanisms (2Bolger G. Michaeli T. Martins T. St. John T. Steiner B. Rodgers L. Riggs M. Wigler M. Ferguson K. Mol. Cell. Biol. 1993; 13: 6558-6571Crossref PubMed Scopus (286) Google Scholar, 4Loughney K. Ferguson K. Schudt C. Dent G. Rabe K.F. The Handbook of Immunopharmacology: Phosphodiesterase Inhibitors. Academic Press, London1996: 1-14Google Scholar). Thus, prolonged increases in cAMP in cells can cause marked increases in the expression of certain PDE4 variants and a PDE-mediated desensitization to the effects of activators of adenylyl cyclase (6Sette C. Iona S. Conti M. J. Biol. Chem. 1994; 269: 9245-9252Abstract Full Text PDF PubMed Google Scholar, 7Alvarez R. Sette C. Yang D. Eglen R. Wilhelm R. Shelton E.R. Conti M. Mol. Pharmacol. 1995; 48: 616-622PubMed Google Scholar, 8Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar, 9Swinnen J.V. Joseph D.R. Conti M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8197-8201Crossref PubMed Scopus (125) Google Scholar, 10Torphy T.J. Zhou H.-L. Cieslinski L.B. J. Pharmacol. Exp. Ther. 1992; 263: 1195-1205PubMed Google Scholar, 11Torphy T.J. Zhou H.-L. Foley J.J. Sarau H.M. Manning C.D. Barnette M.S. J. Biol. Chem. 1995; 270: 23598-23604Crossref PubMed Scopus (91) Google Scholar, 12Verghese M.W. McConnell R.T. Lenhard J.M. Hamacher L. Jin S.-L.C. Mol. Pharmacol. 1995; 47: 1164-1171PubMed Google Scholar, 13Manning C.D. Mclaughlin M.M. Livi G.P. Cieslinski L.B. Torphy T.J. Barnette M.S. J. Pharmacol. Exp. Ther. 1996; 276: 810-818PubMed Google Scholar, 14Erdogan S. Houslay M.D. Biochem. J. 1997; 321: 165-175Crossref PubMed Scopus (114) Google Scholar, 15Rose R.J. Liu H. Palmer D. Maurice D.H. Br. J. Pharmacol. 1997; 122: 233-240Crossref PubMed Scopus (50) Google Scholar, 16Maurice D.H. Cell Biochem. Biophys. 1998; 29: 35-47Crossref PubMed Scopus (8) Google Scholar). Although the cAMP-mediated expression of individual PDE4 genes can be cell type-specific, the generality of this observation and the cellular mechanisms that allow for the selectivity are not known. PDE4 variants are also regulated by protein phosphorylation (reviewed in Ref. 5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar). Thus, a protein kinase A (PKA)-mediated phosphorylation and short-term activation of a specific PDE4 variant, PDE4D3, has been reported (6Sette C. Iona S. Conti M. J. Biol. Chem. 1994; 269: 9245-9252Abstract Full Text PDF PubMed Google Scholar, 7Alvarez R. Sette C. Yang D. Eglen R. Wilhelm R. Shelton E.R. Conti M. Mol. Pharmacol. 1995; 48: 616-622PubMed Google Scholar, 8Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar). Although PKA-mediated phosphorylation was shown to selectively activate a subset of PDE4D3 with high affinity for the inhibitor Rolipram, the molecular basis of this selectivity is unknown at the present time. Phosphorylation of PDE4B2 by mitogen-activated protein kinase (MAPK) has also been reported, although this phosphorylation did not alter the enzymes activity or sensitivity to inhibitors (17Lenhard J.K. Kassel D.B. Rocque W.J. Hamacher L. Holmes W.D. Patel I. Hoffman C. Luther M. Biochem. J. 1996; 316: 751-758Crossref PubMed Scopus (48) Google Scholar). More recently, expression of PDE4A and PDE4D gene products in heterologous expression systems has shown that certain variants of PDE4A and PDE4D are targeted to selected membrane fractions (Ref. 18Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffman R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar and reviewed in Ref. 5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar). The relevance of these findings to the subcellular distribution of PDE4 variants expressed endogenously in nontransformed cells and the impact of selected subcellular expression of these enzymes on their activity and sensitivity to inhibitors have not yet been systematically or rigorously investigated. However, given that targeting of PKA to membrane compartments via specific anchoring proteins (19Scott J.D. Soc. Gen. Physiol. Ser. 1997; 52: 227-239PubMed Google Scholar) has been shown to play a central role on the function of this protein in cells, a similar impact of selective expression of PDE4 variants may perhaps be anticipated. Presumably due to the lack of vasorelaxant effects of PDE4 inhibitor (20Polson J.B. Strada S.J. Ann. Rev. Pharmacol. Toxicol. 1996; 36: 403-427Crossref PubMed Scopus (184) Google Scholar), very few studies have investigated the PDE4 gene products expressed in vascular tissues and the mechanisms regulating their activity and expression. In a previous study, we demonstrated that prolonged activation of adenylyl cyclase in aortic vascular smooth muscle cells (VSMCs) caused up-regulation of total cAMP PDE activity, with at least 60% of this effect due to increases in PDE4 (15Rose R.J. Liu H. Palmer D. Maurice D.H. Br. J. Pharmacol. 1997; 122: 233-240Crossref PubMed Scopus (50) Google Scholar, 16Maurice D.H. Cell Biochem. Biophys. 1998; 29: 35-47Crossref PubMed Scopus (8) Google Scholar). In these earlier studies, we also demonstrated that the cAMP-mediated increase in PDE4 activity was partially responsible for a cAMP-mediated desensitization to activators of adenylyl cyclase in these cells (15Rose R.J. Liu H. Palmer D. Maurice D.H. Br. J. Pharmacol. 1997; 122: 233-240Crossref PubMed Scopus (50) Google Scholar,16Maurice D.H. Cell Biochem. Biophys. 1998; 29: 35-47Crossref PubMed Scopus (8) Google Scholar). More recently, we have reported that PDE4 inhibitors were potent regulators of VSMC migration and could potentiate the effects of inhibitors of other cAMP PDEs on VSMCs (21Palmer D. Tsoi K. Maurice D.H. Circ. Res. 1998; 82: 852-861Crossref PubMed Scopus (80) Google Scholar), a result similar to that previously reported for relaxation of blood vessel (22Komas N. Lugnier C. Stoclet J.-C. Br. J. Pharmacol. 1991; 104: 495-503Crossref PubMed Scopus (132) Google Scholar). In this study, we have investigated the role that protein kinase C (PKC)-mediated activation of the Raf-MEK-ERK cascade plays in regulating the activity and subcellular targeting of the PDE4D variants expressed in VSMCs. Using a combination of pharmacological and biochemical approaches, we have delineated the signaling pathways that allow for the coordinated activation of these PDE4D variants by activators of PKC and PKA. Our data are consistent with a direct role for PKA in activating PDE4D3 and PDE4D5 and a role for PKC, via its effects on the Raf-MEK-ERK cascade, in the activation and translocation of the membrane associated fractions of PDE4D3. Tissue culture reagents (Dulbecco's modified Eagle's medium, calf serum, HEPES, penicillin/streptomycin, Hanks' balanced salt solution (HBSS), and trypsin-EDTA) were from Life Technologies, Inc. Radioactive products (5′-[14C]AMP, [3H]cAMP, and [32P]orthophosphoric acid) were from NEN Life Science Products. Phorbol 12-myristate 13-acetate (PMA), 4α-phorbol-12,13-didecanoate (4αPDD), angiotensin II (AngII), rapamycin, LY 294002, SB202190, Ro 20–1724, Ro-318220, bisindolylmaleimide I, and PD98059 were from Calbiochem-Novachem Corporation, Ontario, Canada. Forskolin, 8-BrcAMP were from Research Biochemicals International (Natick, MA), whereas Tris-HCl, HEPES, benzamidine, EDTA, EGTA, dithiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, and Tween-20 were from ICN Biomedicals Inc. (Costa Mesa, CA). Ionomycin was from Sigma. Leupeptin was from Boehringer Mannheim. Affi-Gel 601 and the column supports were from Bio-Rad. ECL Western blot detection kit was purchased from Amersham Pharmacia Biotech. The BCA protein assay kit and bovine serum albumin were from Pierce. All other chemicals were of reagent grade and purchased from Fisher. PDE4D variants expressed in VSMCs were detected using monoclonal antibody 61D10E. This PDE4D-specific monoclonal antibody was purified from mouse ascites by protein A affinity chromatography monoclonal antibody and supplied to us by Cathy Farrell, Sharon Wolda, and Ken Ferguson of ICOS Corporation (Bothell, WA). Activated ERK1 and ERK2 were detected using a monoclonal antibody that specifically reacts with the phosphorylated forms of these proteins (Promega). Primary cultures of rat aortic VSMCs were established following isolation from rat aortae as described previously and the identity of the VSMCs was confirmed using α-actin staining (15Rose R.J. Liu H. Palmer D. Maurice D.H. Br. J. Pharmacol. 1997; 122: 233-240Crossref PubMed Scopus (50) Google Scholar, 16Maurice D.H. Cell Biochem. Biophys. 1998; 29: 35-47Crossref PubMed Scopus (8) Google Scholar). VSMCs were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 8 mmHEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 95% air-5% CO2 humidified atmosphere. Cells were passaged by washing once with HBSS (without Ca2+ or Mg2+) and then incubating with 1% trypsin-EDTA for 2 min to detach cells and resuspended in growth medium. To maintain cell stocks, 75-cm2 flasks were seeded with 106cells and 20 ml of medium per flask. For all experiments, cells were used between passages 5 and 10. In these experiments, 3 × 105 cells were seeded in 25-cm2 flasks, and the experiments were initiated when the cells reached confluence (3–4 days). Culture medium was removed and replaced with 5.0 ml of fresh culture medium supplemented with either (i) forskolin (0.1–100 μm), (ii) PMA (0.1–1 μm), (iii) angiotensin II (1 μm), (iv) ionomycin (1–100 μm), or (v) vehicle (0.1% dimethyl sulfoxide (Me2SO)) and incubated for various times. After treatment, cells were washed once with 6 ml of HBSS (with Ca2+ and Mg2+) and harvested in 1 ml of lysis buffer containing 50 mm Tris-HCl (pH 7.4), 5 mmMgCl2, 5 mm benzamidine, 1 mm EDTA, 1 mm dithiothreitol, 0.1 mmphenylmethylsulfonyl fluoride, 1 μm leupeptin, and 1% Triton X-100. Cells were removed from the flask by scraping. Cellular debris and unlysed cells were removed by centrifugation at 1000 ×g for 5 min at 4 °C. The 1000 × gsupernatant was transferred to microtubes and stored at 4 °C until assayed for cAMP PDE activity (see below). For some experiments, cytosolic and particulate fractions were prepared. In these instances, VSMCs were lysed as described above, except that Triton X-100 was excluded from the lysis buffer, and the 1000 × gsupernatant was subjected to a further centrifugation at 100,000 × g for 1 h at 4 °C. Cyclic nucleotide phosphodiesterase activity was assayed by a modification of the method of Davis and Daly (23Davis C.W. Daly J.W. J. Cyclic Nucleotide Res. 1979; 5: 65-74PubMed Google Scholar). Reactions were carried out in a total volume of 100 μl containing 5 μmol of Tris-HCl (pH 7.4), 0.5 μmol of MgCl2, 10 nmol of EGTA, and 0.1 nmol of [3H]cAMP (55000–60000 dpm). Following a preincubation period of 2 min at 30 °C, a sample of VSMC homogenate (5 μg of protein) was added, and the reaction was allowed to proceed at 30 °C for 30 min. The reaction was terminated by addition of 50 μl of 0.5m ice-cold EDTA (pH 7.4). Recovery marker (0.1 ml of 5′-[14C]AMP, 1800 dpm) and 0.3 ml of a HEPES-NaCl buffer (0.1 m NaCl, 0.1 m HEPES, pH 8.5) was added to each sample prior to purification of the product of the reaction, 5′-[3H]AMP. 5′-[3H]AMP and 5′-[14C]AMP were recovered by chromatography using a polyacrylamide-boronate gel column (Affi-Gel 601, Bio-Rad; bed volume, 1 ml). Samples were applied following prewashing of the columns with 8 ml of HEPES-NaCl buffer. After four additional washes of the columns with 2 ml of HEPES-NaCl and equilibration of the columns with 1 ml of 0.05 m sodium acetate (pH 4.8), the 5′-AMP was eluted with 4 ml of 0.05 m sodium acetate. The recovered 5′-[3H]AMP was quantified by liquid scintillation counting, corrected for recovery of 5′-[14C]AMP, and normalized to the total protein used in the assay, and the total activity was expressed as pmol min−1 mg−1 of protein. Rat aortic VSMC cultures incubated with compounds of interest were homogenized in a buffer consisting of 20 mm Tris-HCl (pH 7.5), 1 mm MgCl2, 0.1 mm EGTA, 5 mm benzamidine, 1 μg/ml aprotinin, and 1 μg/ml leupeptin (Buffer A). These samples (5–20 μg of total homogenate protein or of protein from isolated subcellular fractions) were subjected to SDS-PAGE. Following electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad), and the membranes were blocked by incubation with TBST (20 mm Tris, pH 7.5, 100 mm NaCl, 0.1% Tween-20) supplemented with 5% powdered nonfat milk for 1 h. Blots were incubated with an appropriate dilution of primary antibodies for 1–2 h and rinsed three times with TBST. Rinsed blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG for 1 h, then rinsed with TBST, and immunoreactivity was detected by chemiluminescence as per the manufacturer's recommendations (Amersham Pharmacia Biotech). Rat aortic VSMCs were seeded in T75 boxes (Corning) and cultured as described above. At confluence, the growth medium was replaced with phosphate-free minimal essential medium containing 20 mm HEPES (pH 7.4) and carrier-free [32P]orthophosphate (0.2 mCi/ml), and cells were incubated for 2 h. During the last 0.5 h of the metabolic labeling, PMA (100 nm), FSK (100 μm), or Me2SO was added. At the end of this treatment, cells were washed twice with HBSS; harvested in a buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.1% SDS, 100 μg of phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate; and homogenized. After centrifugation at 1000 × g for 5 min, supernatants were incubated batch-wise for 30 min with DEAE-Sepharose buffered with 200 mm sodium acetate, pH 6.5. The resin was washed twice with the same buffer, and the adsorbed proteins were eluted with 600 mm sodium acetate, pH 6.5. In order to remove nonspecific immunoreactive proteins, the partially purified samples were incubated with 2.5 μg/ml of an irrelevant monoclonal antibody (anti-elastase, mouse IgG2b) for 2 h at 4 °C and then with protein G beads (Amersham Pharmacia Biotech) for 30 min and centrifuged (1000 ×g for 15 min). The supernatant were transferred to fresh vials and incubated with 2.5 μg/ml of the PDE4D-specific monoclonal antibody (61D10E) for 16 h at 4 °C. Again, precipitation was achieved using protein G beads. The immunoprecipitates were washed five times and resuspended in 1% SDS in phosphate-buffered saline, diluted in SDS-PAGE sample buffer, and separated by 7.5% SDS-PAGE. Proteins were blotted onto an Immobilon membrane, and the radioactive bands were visualized and quantitated with an Instant Imager (Packard Instrument Co.). Protein was determined using the BCA protein assay system from Pierce, according to the manufacturer's protocol, with bovine serum albumin as the standard. Numerical data (PDE activities) and densitometric determinations of PDE4 phosphorylation status are presented as mean ± S.E. of at least three independent experiments. Immunoblots that are shown are representative of results obtained in at least three individual experiments. Statistical differences between cAMP PDE activities were determined using the Student's t test for either paired or unpaired samples, with p < 0.05 considered significant. Incubation of cultured VSMCs with the activator of adenylyl cyclase, forskolin, caused a time- and concentration-dependent increase in cAMP PDE activity (Table I). Similarly, incubation of these cells with PMA or with the vasoactive agent AngII caused time and concentration dependent increases in cAMP PDE activity in these cells, although the absolute magnitude of the increase brought about with these agents was less marked than was obtained with forskolin (TableI). Coincubation of PMA or AngII with forskolin resulted in an additive increase in cAMP PDE activity when compared with the effects of the individual agents used alone (Table I). The inactive analogues of forskolin, 1,9-dideoxyforskolin, and PMA, 4α-phorbol-12,13-didecanoate (4αPDD), had no effect on total cAMP PDE activity or on the fraction attributable to PDE4 (not shown). As reported previously, PDE4 activity accounts for approximately 60% of total cAMP PDE activity in these cells (15Rose R.J. Liu H. Palmer D. Maurice D.H. Br. J. Pharmacol. 1997; 122: 233-240Crossref PubMed Scopus (50) Google Scholar), and based on the effects of selective PDE4 inhibition, increases in this fraction of the total activity accounted for more than 90% of the total increase under our experimental conditions (Table I). The increases in PDE4 activity caused by either forskolin, PMA, or AngII or combinations of these agents were not sensitive to cycloheximide or actinomycin D.Table IIncubation of VSMC with forskolin, PMA, and AngII increases PDE4 activityAdditionscAMP PDEPDE3PDE4ΔPDE4pmol/min/mg%None70.6 ± 1.210.6 ± 1.945.9 ± 0.5Forskolin (1.0 μm)72.9 ± 0.8aP < 0.05 in comparison to no addition value.11.3 ± 1.347.8 ± 0.94Forskolin (10 μm)85.3 ± 2.0aP < 0.05 in comparison to no addition value.14.3 ± 1.156.9 ± 1.8aP < 0.05 in comparison to no addition value.24Forskolin (100 μm)90.1 ± 2.9aP < 0.05 in comparison to no addition value.12.9 ± 2.467.3 ± 2.0aP < 0.05 in comparison to no addition value.47PMA (1.0 nm)71.3 ± 1.010.9 ± 1.446.3 ± 0.91PMA (10 nm)74.3 ± 1.511.3 ± 2.747.9 ± 1.24PMA (100 nm)80.9 ± 1.7aP < 0.05 in comparison to no addition value.9.8 ± 2.051.5 ± 1.7aP < 0.05 in comparison to no addition value.12AngII (10 nm)77.2 ± 0.9aP < 0.05 in comparison to no addition value.11.7 ± 1.349.9 ± 0.8aP < 0.05 in comparison to no addition value.9AngII (100 nm)83.5 ± 1.3aP < 0.05 in comparison to no addition value.14.9 ± 2.253.6 ± 1.1aP < 0.05 in comparison to no addition value.17Forskolin (100 μm) + PMA (100 nm)115.0 ± 2.3aP < 0.05 in comparison to no addition value.14.8 ± 2.074.9 ± 2.1aP < 0.05 in comparison to no addition value.63Forskolin (100 μm) + AngII (100 nm)113.9 ± 2.9aP < 0.05 in comparison to no addition value.13.7 ± 2.884.9 ± 8.8aP < 0.05 in comparison to no addition value.85VSMCs were incubated with forskolin (100 μm), PMA (100 nm), or AngII (100 nm) for 30 min. Following the incubation, cells were rinsed with HBSS, homogenized in lysis buffer containing 1% Triton X100, and centrifuged at 1000 ×g for 10 min. PDE activity was determined as described under “Experimental Procedures,” and cilostamide (1 μm) or Ro, 20–1724 (10 μm) was used to determine PDE3 and PDE4 activities, respectively. Values are mean ± S.E. from four determinations.a P < 0.05 in comparison to no addition value. Open table in a new tab VSMCs were incubated with forskolin (100 μm), PMA (100 nm), or AngII (100 nm) for 30 min. Following the incubation, cells were rinsed with HBSS, homogenized in lysis buffer containing 1% Triton X100, and centrifuged at 1000 ×g for 10 min. PDE activity was determined as described under “Experimental Procedures,” and cilostamide (1 μm) or Ro, 20–1724 (10 μm) was used to determine PDE3 and PDE4 activities, respectively. Values are mean ± S.E. from four determinations. Because incubation of VSMCs with forskolin, PMA, or AngII increased PDE4 activity, and cAMP-mediated, PKA-dependent activation of a PDE4 variant (PDE4D3) had previously been reported (6Sette C. Iona S. Conti M. J. Biol. Chem. 1994; 269: 9245-9252Abstract Full Text PDF PubMed Google Scholar, 7Alvarez R. Sette C. Yang D. Eglen R. Wilhelm R. Shelton E.R. Conti M. Mol. Pharmacol. 1995; 48: 616-622PubMed Google Scholar, 8Sette C. Conti M. J. Biol. Chem. 1996; 271: 16526-16534Crossref PubMed Scopus (357) Google Scholar), we undertook to determine which PDE4D gene products were expressed in these cells and determine whether phosphorylation of these enzymes was involved in the increased PDE4 activity caused by forskolin, PMA, AngII, or combinations of these compounds. Immunoblot analysis of lysates of VSMCs with a PDE4D-selective monoclonal antibody identified two individual anti-PDE4D immunoreactive species with electrophoretic mobilities by SDS-PAGE characteristic of previously described PDE4D variants (Fig.1). The smaller more abundant PDE4D species migrated with an observed molecular mass of 95 ± 2 kDa by SDS-PAGE, a size similar to that of the human recombinant PDE4D3 (Fig.1). Based on these characteristics, we identified this protein as PDE4D3. The larger, less abundant PDE4 species migrated with an observed molecular mass of 105 ± 3 kDa (Fig. 1), a mobility consistent with PDE4D5 (18Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffman R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar). Because this immunoreactive protein was significantly smaller than the other known PDE4D species, PDE4D4, which has been shown to migrate with an observed molecular mass of approximately 119 kDa (18Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffman R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar), we identified the larger VSMC PDE4D variant as PDE4D5 (5Houslay M.D. Sullivan M. Bolger G.B. August T.J. Murad F. Anders M.W. Coyle J.T. Advances in Pharmacology. 44. Academic Press, London1998: 225-342Google Scholar, 18Bolger G.B. Erdogan S. Jones R.E. Loughney K. Scotland G. Hoffman R. Wilkinson I. Farrell C. Houslay M.D. Biochem. J. 1997; 328: 539-548Crossref PubMed Scopus (173) Google Scholar). Consistent with this identification, polymerase chain reactions carried out using oligo(dT)18-primed VSMC mRNA as a template and PDE4D3- and PDE4D5-specific oligonucleotide primers allows the amplification of both PDE4D3 and PDE4D5 (not shown). When cells were lysed in the absence of detergent, PDE4D3 was found primarily in the supernatant fraction, with approximately 17% present in the 100,000 × g pellets (Fig. 1). In contrast, PDE4D5 was present in both the supernatant and particulate fractions in roughly equal amounts (Fig. 1). Although the amount of PDE4D5 detected in our experiments was variable, it was never present at more than 15% of the level of PDE4D3. The variability in the levels of PDE4D5 detected in our studies may be related to our observation that the fraction of PDE4D5 expressed in the particulate fraction was not efficiently solubilized with Triton X-100, the detergent used in most of our studies (not shown). In most previous reports, phosphorylation of PDE4D3 has been studied by quantitating the fraction of PDE4D3 that exhibited a retarded migration by SDS-PAGE, an event that is detected as an upward shift in the electrophoretic mobility of PDE4D3. Using this approach, all of our d" @default.
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