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• W2024298712 abstract "Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation. Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation. Mitochondria play central roles in cellular energy metabolism, as well as in the regulation of cell cycle progression, apoptosis, and aging processes (1Balaban R.S. Nemoto S. Finkel T. Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3165) Google Scholar, 2Goldenthal M.J. Marín-García J. Mol. Cell. Biochem. 2004; 262: 1-16Crossref PubMed Scopus (158) Google Scholar). Despite their importance, signaling into, from, and within mitochondria is still not well understood. Emerging signaling mechanisms in mitochondria and between the organelle and its environment include reversible protein deacetylation (3Schlicker C. Hall R.A. Vullo D. Middelhaufe S. Gertz M. Supuran C.T. Mühlschlegel F.A. Steegborn C. J. Mol. Biol. 2009; 385: 1207-1220Crossref PubMed Scopus (177) Google Scholar, 4Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1194) Google Scholar), redox regulation and reactive oxygen species formation (5Gertz M. Fischer F. Wolters D. Steegborn C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5705-5709Crossref PubMed Scopus (66) Google Scholar, 6Gertz M. Steegborn C. Antioxid. Redox Signal. 2010; 13: 1417-1428Crossref PubMed Scopus (66) Google Scholar, 7Paulsen C.E. Carroll K.S. ACS Chem. Biol. 2010; 5: 47-62Crossref PubMed Scopus (387) Google Scholar), and cyclic adenosine monophosphate (cAMP) signaling (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 9Lakshminarasimhan M. Steegborn C. Exp. Gerontol. 2011; 46: 174-177Crossref PubMed Scopus (26) Google Scholar). cAMP-dependent effects and proteins of cAMP signaling systems, such as cAMP-responsive element-binding protein (CREB), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs), 3The abbreviations used are: AKAPA-kinase anchoring proteinCOXcytochrome c oxidaseEHNAerythro-9-(2-hydroxyl-3-nonyl)adeninePDEphosphodiesterasePKproteinase KMapmitochondrial associated proteinSACsoluble adenylyl cyclaseICQintensity correlation quotientIBMX3-isobutyl-1-methylxanthin8-Br-cAMP8-bromoadenosine-3′, 5′-cyclic monophosphateRrPearson's correlation coefficient. have been described in mitochondria (10Sardanelli A.M. Signorile A. Nuzzi R. Rasmo D.D. Technikova-Dobrova Z. Drahota Z. Occhiello A. Pica A. Papa S. FEBS Lett. 2006; 580: 5690-5696Crossref PubMed Scopus (70) Google Scholar, 11Cammarota M. Paratcha G. Bevilaqua L.R. Levi de Stein M. Lopez M. Pellegrino de Iraldi A. Izquierdo I. Medina J.H. J. Neurochem. 1999; 72: 2272-2277Crossref PubMed Scopus (81) Google Scholar, 12Ryu H. Lee J. Impey S. Ratan R.R. Ferrante R.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 13915-13920Crossref PubMed Scopus (133) Google Scholar). In addition to these effector proteins, a complete cAMP signaling microdomain requires enzymes for synthesis and degradation of the second messenger. Although an intramitochondrial cAMP source has been identified recently (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), there is no known cAMP-degrading enzyme in this organelle. Cyclic AMP is formed inside mitochondria by soluble adenylyl cyclase (sAC) (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), a member of Class III of the nucleotidyl cyclase family, which also comprises the G-protein-regulated transmembrane adenylyl cyclases (13Kamenetsky M. Middelhaufe S. Bank E.M. Levin L.R. Buck J. Steegborn C. J. Mol. Biol. 2006; 362: 623-639Crossref PubMed Scopus (242) Google Scholar). Unique from transmembrane adenylyl cyclases, sAC is activated by bicarbonate (14Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Science. 2000; 289: 625-628Crossref PubMed Scopus (669) Google Scholar), and it appears to act as a metabolic sensor (15Zippin J.H. Levin L.R. Buck J. Trends Endocrinol. Metab. 2001; 12: 366-370Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), whose mitochondrial form(s) seems to modulate PKA-mediated regulation of respiration (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar) and apoptosis (16Kumar S. Kostin S. Flacke J.P. Reusch H.P. Ladilov Y. J. Biol. Chem. 2009; 284: 14760-14768Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A-kinase anchoring protein cytochrome c oxidase erythro-9-(2-hydroxyl-3-nonyl)adenine phosphodiesterase proteinase K mitochondrial associated protein soluble adenylyl cyclase intensity correlation quotient 3-isobutyl-1-methylxanthin 8-bromoadenosine-3′, 5′-cyclic monophosphate Pearson's correlation coefficient. The opponents of the cyclic nucleotide-forming cyclases are cyclic nucleotide monophosphate (cNMP)-degrading phosphodiesterases (PDEs). Mammalian cells contain a varying subset of members of the classical PDE family, which comprises 11 PDE gene families (PDE1–11) (17Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (930) Google Scholar, 18Omori K. Kotera J. Circ. Res. 2007; 100: 309-327Crossref PubMed Scopus (562) Google Scholar) and non-generic PDEs such as the protein human Prune (19Middelhaufe S. Garzia L. Ohndorf U.M. Kachholz B. Zollo M. Steegborn C. Biochem. J. 2007; 407: 199-205Crossref PubMed Scopus (14) Google Scholar, 20D'Angelo A. Garzia L. André A. Carotenuto P. Aglio V. Guardiola O. Arrigoni G. Cossu A. Palmieri G. Aravind L. Zollo M. Cancer Cell. 2004; 5: 137-149Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The isoforms of the generic PDEs comprise homologous catalytic domains, fused to varying regulatory domains, making them sensitive to a variety of signals such as calmodulin or cNMP binding (17Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (930) Google Scholar). This regulated degradation of cNMPs, together with their regulated synthesis, determines local cNMP concentrations and thus signal strength and duration. PDE1, for example, contributes to formation of cytosolic and possibly nuclear cNMP signals that can be modulated by Ca2+/calmodulin (18Omori K. Kotera J. Circ. Res. 2007; 100: 309-327Crossref PubMed Scopus (562) Google Scholar), and the cGMP-activated, specifically cGMP-hydrolyzing PDE5 can contribute to the formation of cGMP signal spikes in platelets (21Mullershausen F. Russwurm M. Thompson W.J. Liu L. Koesling D. Friebe A. J. Cell Biol. 2001; 155: 271-278Crossref PubMed Scopus (110) Google Scholar). Most PDE genes encode several splice variants, such as PDE2A1, PDE2A2, and PDE2A3 (22Rosman G.J. Martins T.J. Sonnenburg W.K. Beavo J.A. Ferguson K. Loughney K. Gene. 1997; 191: 89-95Crossref PubMed Scopus (112) Google Scholar, 23Sonnenburg W.K. Mullaney P.J. Beavo J.A. J. Biol. Chem. 1991; 266: 17655-17661Abstract Full Text PDF PubMed Google Scholar, 24Yang Q. Paskind M. Bolger G. Thompson W.J. Repaske D.R. Cutler L.S. Epstein P.M. Biochem. Biophys. Res. Commun. 1994; 205: 1850-1858Crossref PubMed Scopus (66) Google Scholar), resulting in more than 100 differently expressed PDEs, which can further contribute to differential modulation and localization of cNMP signals (17Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (930) Google Scholar). Although PDE activity has been observed in mitochondria (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), the identity of the mitochondrial PDE remained unknown. Such a mitochondrial PDE should reside inside the matrix to ensure that local sAC-generated cAMP signals are temporally and spatially controlled. PDE4 co-localization with mitochondria, apparently through an interaction with the protein “disrupted in schizophrenia 1” (DISC1), was previously reported (25Millar J.K. Pickard B.S. Mackie S. James R. Christie S. Buchanan S.R. Malloy M.P. Chubb J.E. Huston E. Baillie G.S. Thomson P.A. Hill E.V. Brandon N.J. Rain J.C. Camargo L.M. Whiting P.J. Houslay M.D. Blackwood D.H. Muir W.J. Porteous D.J. Science. 2005; 310: 1187-1191Crossref PubMed Scopus (560) Google Scholar), but a localization of this, or any other, PDE inside mitochondria has not been shown. Identifying mitochondrial PDEs would reveal specifically localized, well “druggable” targets for treatment of mitochondrial diseases (26Acin-Perez R. Salazar E. Brosel S. Yang H. Schon E.A. Manfredi G. EMBO Mol. Med. 2009; 1: 392-406Crossref PubMed Scopus (89) Google Scholar) because PDEs are established as excellent targets for pharmacological inhibition. Specific compounds are available for most isoforms, including clinically used drugs for PDE3 and PDE5, and several isoforms serve as therapeutic targets in current drug development efforts (27Jeon Y.H. Heo Y.S. Kim C.M. Hyun Y.L. Lee T.G. Ro S. Cho J.M. Cell. Mol. Life Sci. 2005; 62: 1198-1220Crossref PubMed Scopus (200) Google Scholar). Here, we show that PDE2A isoform 2 is specifically targeted to the mitochondrial matrix, where it forms a local signaling system with sAC regulating the respiratory chain. Our findings complete the cAMP signaling system in mitochondria and have implications for understanding and pharmacologically targeting mitochondrial processes and specific PDE isoforms. For the import experiments and initial characterization of lysates in PDE activity and inhibition assays, mitochondria from rat liver and brain were enriched using differential centrifugation. Homogenized liver or brain cells in ice-cold isolation buffer (2 mm Hepes, pH 7.4, 220 mm mannitol, and 70 mm sucrose) were disrupted mechanically. To remove cell debris and nuclei, lysates were centrifuged at 500 × g for 3 min at 4 °C. The supernatant was centrifuged two more times. Mitochondria were collected by centrifugation at 26,000 × g for 10 min at 4 °C. The pellet was washed in isolation buffer and recentrifuged twice, and the mitochondria were finally resuspended in 2 ml of isolation buffer. For Western blot analysis and functional assays on mitochondria, highly pure mitochondria were prepared from mouse liver and brain mitochondria as described (28Fernández-Vizarra E. López-Pérez M.J. Enriquez J.A. Methods. 2002; 26: 292-297Crossref PubMed Scopus (133) Google Scholar). Isolated mitochondria were incubated with sAC or PDE inhibitors in MAITE medium (10 mm Tris-HCl, pH 7.4; 25 mm sucrose; 75 mm sorbitol; 100 mm KCl; 10 mm K2HPO4; 0.05 mm EDTA; 5 mm MgCl2; 1 mg/ml BSA) in the presence of a mixture of phosphatase inhibitors (Sigma-Aldrich). The following conditions were used for all experiments: 1 mm 8Br-cAMP (Sigma-Aldrich), 15 μm erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA) (Tocris), 20 nm BAY60-7550 (Tocris), 1 μm rolipram (Sigma-Aldrich), and 25 μm KH7 for 10 min. For protein localization, pure brain mitochondria were isolated using a Percoll gradient as described previously (29Fischer L.R. Igoudjil A. Magrané J. Li Y. Hansen J.M. Manfredi G. Glass J.D. Brain. 2011; 134: 196-209Crossref PubMed Scopus (86) Google Scholar). 500 μg of mitochondria (10 mg/ml) were resuspended in MS-EGTA (225 mm mannitol, 75 mm sucrose, 5 mm HEPES, 1 mm EGTA, pH 7.4). Water (1/10 volume) and digitonin (1 mg of digitonin/5 mg of mitochondrial protein) were added, and the mixture was incubated on ice for 45 min. Then, KCl (150 mm) was added followed by incubation for 2 min on ice and centrifugation at 18,000 × g for 20 min at 4 °C. The pellet containing the mitoplast fraction was resuspended at 1 mg/ml in 300 mm Tris-HCl, 10 μm CaCl2, pH 7.4. The supernatant containing the post-mitoplast fraction was precipitated with 12% TCA and centrifuged at 18,000 × g for 15 min at 4 °C. The pellet was resuspended in 500 μl of acetone and centrifuged at 18,000 × g for 15 min at 4 °C. For protease protection assays, 20 μg of mitochondrial protein were treated with 20 μg/ml proteinase K (PK) for 20 min on ice. Then, PK was inactivated with 2 mm phenylmethanesulfonyl fluoride (PMSF) for 10 min on ice. Prior to PK treatment, one aliquot of mitochondria was solubilized with 1% Triton X-100 (Sigma-Aldrich) for 15 min on ice. For Western blot analyses of mitochondria and mitoplast samples, 25 μg of protein were separated by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto PVDF filters (Bio-Rad). For protein detection, the following antibodies were used: cytochrome c oxidase (COX) subunit I (Invitrogen), Hsp60, grp75, and cytochrome c (StressGen); PDE2A (K-20, Cell Signaling); Tim23 (BD Transduction Laboratories); and β-actin (Sigma-Aldrich). For the characterization of the PDE activity in mitochondrial lysates, cAMP- and cGMP-degrading activities were determined in a radioactive assay, using 10 μm [32P]cAMP or [32P]cGMP as substrate and 10-min incubations, as described before (30Jäger R. Schwede F. Genieser H.G. Koesling D. Russwurm M. Br. J. Pharmacol. 2010; 161: 1645-1660Crossref PubMed Scopus (24) Google Scholar). Measurements of mitochondrial cAMP levels after treatment of whole organelles were performed according to manufacturer's instructions using the Direct Correlate-EIA cAMP kit (Assay Designs Inc.). 100 μl of sample were measured. If necessary, samples were diluted to bring the cAMP level of the sample within the linear range of the assay. For the in vitro test, fragments coding for PDE2A2(1–210) and PDE2A2(18–210) of mouse PDE2A2 (NM_001143849.1, designated as transcript variant/isoform 3 in contrast to the original designation PDE2A2 (gene identifier 706929), see Refs. 24Yang Q. Paskind M. Bolger G. Thompson W.J. Repaske D.R. Cutler L.S. Epstein P.M. Biochem. Biophys. Res. Commun. 1994; 205: 1850-1858Crossref PubMed Scopus (66) Google Scholar, 31Bender A.T. Beavo J.A. Pharmacol. Rev. 2006; 58: 488-520Crossref PubMed Scopus (1383) Google Scholar, and 32Juilfs D.M. Soderling S. Burns F. Beavo J.A. Rev. Physiol Biochem. Pharmacol. 1999; 135: 67-104Crossref PubMed Google Scholar) were cloned using BamHI and XhoI into pCDNA3.1zeo(+) with two additional methionine residues at the C terminus. For cloning PDE(1–17)-Map(45–203), an additional SacII restriction site was introduced into PDE2A2 by site-directed mutagenesis, resulting in an insertion encoding an additional proline and arginine. After restriction, the fragment for the PDE2A2 N terminus was ligated into a cleaved pCDNA3.1zeo(+)-Map(45–203) plasmid. Radiolabeled proteins were synthesized in reticulocyte lysate (TnT T7 coupled reticulocyte lysate system, Promega) containing [35S]Met. Rat liver mitochondria were isolated as described before (33Domańska G. Motz C. Meinecke M. Harsman A. Papatheodorou P. Reljic B. Dian-Lothrop E.A. Galmiche A. Kepp O. Becker L. Günnewig K. Wagner R. Rassow J. PLoS Pathog. 2010; 6: e1000878Crossref PubMed Scopus (33) Google Scholar). The mitochondria (30 μg of mitochondrial protein) were incubated with 5 μl of reticulocyte lysate in 50 μl of 250 mm sucrose, 80 mm KCl, 20 mm potassium phosphate, 2 mm NADH, 2 mm ATP, 10 mm MOPS/KOH, pH 7.2, containing 3% (w/v) bovine serum albumin. After incubation at 25 °C for 10 min, the mitochondria were reisolated by centrifugation (10 min 16,000 × g). To remove residual reticulocyte lysate, the mitochondria were resuspended in 100 μl of 250 mm sucrose, 150 mm NaCl, 10 mm MOPS/KOH, pH 7.2, and again reisolated. Mitochondria-associated proteins were separated by SDS-PAGE and analyzed using a BAS-1800 II imager (Fujifilm). For the in vivo localization studies, the gene fragment coding for the N terminus (amino acids 1–216) of PDE2A isoform 2 (NM_001143849.1, see above) was cloned using EcoRI and BamHI into pEGFP-N3 (Clontech) to yield a construct with C-terminally fused GFP. For comparison, we used similar constructs for PDE2A isoforms 1 and 3 fused to CFP as described before (34Russwurm C. Zoidl G. Koesling D. Russwurm M. J. Biol. Chem. 2009; 284: 25782-25790Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For expression, each PDE fusion construct was co-transfected with pDsRed-mito (Clontech), which codes for a fusion of Dicosoma sp. red fluorescent protein (DsRed) with the mitochondrial targeting sequence of human cytochrome c oxidase subunit VIII, into HEK-293 cells using FuGENE 6 (Roche Applied Science) and standard procedures. PDE isoform constructs and DsRed-mito were expressed under control of the immediate early promoter of cytomegalovirus, and cells from three independent expression experiments were visualized using a Zeiss LSM 510 Meta system and a Plan-Neofluar 100×/1.3 objective in sequential scan mode using the 458 (CFP), 488 (EGFP), and 564 nm (DsRed) laser lines with corresponding band-path filters. Image recordings were optimized using the LSM 510 software with images representing single focal planes with a pixel resolution of 0.09 × 0.09 μm. Images were imported into ImageJ, background was subtracted, and regions of interest representing >100 cells for each condition were subjected to intensity correlation analysis (35Li Q. Lau A. Morris T.J. Guo L. Fordyce C.B. Stanley E.F. J. Neurosci. 2004; 24: 4070-4081Crossref PubMed Scopus (568) Google Scholar). Pearson's correlation coefficients were calculated with a value of 1 representing perfect correlation; −1 represents perfect exclusion, and zero represents random localization. Because values close to zero have to be critically evaluated, we calculated in addition the intensity correlation quotient (ICQ) with random staining: ICQ ∼0; segregated staining: 0 > ICQ ≥ −0.5; dependent staining: 0 < ICQ ≤ +0.5. Significance values were calculated using an unpaired Student's t test with p > 0.01. Oligomycin-sensitive mitochondrial ATP synthesis was measured in isolated mitochondria (15–25 μg of protein) in the presence of ADP (0.1 mm) and malate plus pyruvate (1 mm each) as substrates and of the adenylate kinase inhibitor diadenosine pentaphosphate (0.15 mm), using a kinetic luciferase-luciferin detection system in a recording luminometer, as described previously (36Vives-Bauza C. Yang L. Manfredi G. Methods Cell Biol. 2007; 80: 155-171Crossref PubMed Scopus (82) Google Scholar). These conditions yielded linear ATP synthesis rates, which were expressed as rate (nmol/min) of ATP production/mg of mitochondrial protein. Pyruvate/malate-driven oxygen consumption was measured in isolated mitochondria using an Oxygraph system equipped with a Clark electrode (Hansatech; 100 μg of protein) in the presence of ADP, as described previously (37Hofhaus G. Shakeley R.M. Attardi G. Methods Enzymol. 1996; 264: 476-483Crossref PubMed Google Scholar). Mitochondrial respiration was expressed as μmol of O2 consumed/min/mg of mitochondrial protein. COX activity in isolated mitochondria (2–5 μg of mitochondrial protein) was measured spectrophotometrically as described (38Birch-Machin M.A. Turnbull D.M. Methods Cell Biol. 2001; 65: 97-117Crossref PubMed Google Scholar). We recently showed that sAC-dependent cAMP formation in the matrix of liver mitochondria regulates respiration (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). We now demonstrate that cAMP also increases ATP production in mitochondria isolated from brain via stimulation of the electron transport chain (Fig. 1A). This form of regulation thus exists in liver and brain, and we assume that it will prove to be a general mitochondrial mechanism. We also showed that cAMP does not pass through the inner mitochondrial membrane (8Acin-Perez R. Salazar E. Kamenetsky M. Buck J. Levin L.R. Manfredi G. Cell Metab. 2009; 9: 265-276Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar); thus, this signaling pathway requires a mitochondrial cAMP-degrading PDE for switching off the signal. As a first step toward identifying candidate isoforms for such a mitochondrial PDE, we analyzed lysates of isolated mitochondria from rat liver and brain for cAMP- and cGMP-degrading activities in the absence and presence of different PDE inhibitors (Fig. 1, B and C). Mitochondrial homogenates from both tissues contained readily detectable cAMP- and cGMP-degrading activities. The cAMP-degrading mitochondrial activities from liver and brain were inhibited by ∼50 and 70%, respectively, by the non-selective PDE inhibitor 3-isobutyl-1-methylxanthin (IBMX; Fig. 1B). In contrast, cGMP-degrading activity found in mitochondria from both tissues was almost completely inhibited by this non-selective PDE inhibitor (Fig. 1C). To further characterize the PDE isoforms responsible for these activities, isoform-specific inhibitors of PDEs 1 through 5 were employed. The inhibitors of PDE1 (vinpocetine), PDE3 (milrinone), and PDE5 (sildenafil) had little to no effect on cAMP- or cGMP-degrading activities in mitochondrial homogenates from either tissue (Fig. 1, B and 1C). In contrast, the inhibitor BAY60-7550 (“BAY60”), which is specific for PDE2A, a PDE that can degrade both cAMP and cGMP, inhibited the cAMP-degrading activities significantly (Fig. 1B). BAY60 inhibited the activity from liver mitochondria to an extent comparable with IBMX and inhibited activity from brain mitochondria slightly less than IBMX. To evaluate the specificity of BAY60, concentration-response curves for inhibition of mitochondrial cAMP-degrading activity by this compound were recorded (Fig. 1D). The obtained IC50 of ∼10 nm for the BAY60-sensitive activity is in good agreement with the IC50 for BAY60 inhibition of PDE2 (4.7 nm (39Boess F.G. Hendrix M. van der Staay F.J. Erb C. Schreiber R. van Staveren W. de Vente J. Prickaerts J. Blokland A. Koenig G. Neuropharmacology. 2004; 47: 1081-1092Crossref PubMed Scopus (260) Google Scholar)) and confirms specificity of the BAY60 effect. BAY60 also inhibited almost all cGMP-degrading activity present in mitochondrial homogenates from both tissues, comparable with the nonspecific inhibitor IBMX (Fig. 1C). Thus, PDE2A appears to be present in brain and liver mitochondrial fractions and to be responsible for all (liver) or most (brain) of the IBMX-sensitive PDE activity in mitochondria from these tissues. Rolipram, the inhibitor of PDE4, had no significant effect on cAMP-degrading activity from liver mitochondria but showed some inhibition of the activity from brain, albeit to a lesser extent than BAY60 (Fig. 1B). Thus, lysates from brain mitochondria seem to contain PDE4 in addition to PDE2A, and IBMX inhibition of brain mitochondrial PDE activity seems to correspond to the sum of the effects of the PDE4-selective rolipram and the PDE2A-selective BAY60. To further substantiate the identity of the mitochondrial IBMX-sensitive PDE as PDE2A, stimulation of this activity by cGMP was analyzed (Fig. 1E). In accordance with its known effects on PDE2 (40Beavo J.A. Hardman J.G. Sutherland E.W. J. Biol. Chem. 1971; 246: 3841-3846Abstract Full Text PDF PubMed Google Scholar, 41Yamamoto T. Manganiello V.C. Vaughan M. J. Biol. Chem. 1983; 258: 12526-12533Abstract Full Text PDF PubMed Google Scholar), cGMP stimulated cAMP degradation with an EC50 of ∼0.3 μm, and it inhibited at higher concentrations. cGMP stimulated cAMP degradation 2–3-fold, which is lower than reported (40Beavo J.A. Hardman J.G. Sutherland E.W. J. Biol. Chem. 1971; 246: 3841-3846Abstract Full Text PDF PubMed Google Scholar, 41Yamamoto T. Manganiello V.C. Vaughan M. J. Biol. Chem. 1983; 258: 12526-12533Abstract Full Text PDF PubMed Google Scholar) and might reflect the presence of an additional cAMP-degrading PDE, as suggested by the incomplete suppression of cAMP-degrading activity by IBMX or BAY60 described above. In conclusion, our results strongly suggest that brain and liver mitochondria contain PDE2A and a yet to be identified, IBMX-insensitive, cAMP-specific PDE (see “Discussion”). To confirm the presence of PDE2A in mitochondria and to elucidate the PDE2-containing subcompartment, we tested highly purified mitochondria and fractionated mitochondrial subcompartments with a PDE2A-specific antibody. Western blots of whole mitochondria isolated from liver and brain tissue showed positive bands of the expected molecular mass (∼105 kDa; Fig. 2A). Tissue homogenates from brain produced a stronger signal than those from liver, consistent with the previously demonstrated prominent expression of PDE2A in brain (22Rosman G.J. Martins T.J. Sonnenburg W.K. Beavo J.A. Ferguson K. Loughney K. Gene. 1997; 191: 89-95Crossref PubMed Scopus (112) Google Scholar, 34Russwurm C. Zoidl G. Koesling D. Russwurm M. J. Biol. Chem. 2009; 284: 25782-25790Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 42Repaske D.R. Corbin J.G. Conti M. Goy M.F. Neuroscience. 1993; 56: 673-686Crossref PubMed Scopus (88) Google Scholar); however, the PDE2A-specific signal in mitochondria from brain and liver was more comparable, revealing that differences in the levels of the mitochondrially localized PDE2A population are less pronounced. We next determined which mitochondrial subcompartment contains PDE2A using pure mitochondria from brain. Our Western blots revealed PDE2A in mitochondrial fractions and in the mitoplast fractions, but not in lysates after mitoplast removal (post-mitoplast; Fig. 2B). Furthermore, PDE2A was protected during proteinase K treatment of mitoplasts, as were hsp60 and grp75, two mitochondrial matrix proteins (Fig. 2C). Because post-mitoplast supernatants contain intermembrane space and outer membrane proteins and proteinase K treatment would digest accessible proteins, we conclude that PDE2A is enclosed in the mitochondrial matrix. Consistently, PDE2A was not protected from proteinase K digestion anymore when the mitoplast membrane (inner mitochondrial membrane) was solubilized through the addition of Triton (Fig. 2C). Thus, PDE2A is co-localized with the cAMP source sAC in the mitochondrial matrix. Mitochondrial transport systems can import proteins that contain different types of localization signals, but most matrix proteins contain an N-terminal sorting signal, which is often proteolytically removed during import (43Chacinska A. Koehler C.M. Milenkovic D. Lithgow T. Pfanner N. Cell. 2009; 138: 628-644Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 44Vögtle F.N. Wortelkamp S. Zahedi R.P. Becker D. Leidhold C. Gevaert K. Kellermann J. Voos W. Sickmann A. Pfanner N. Meisinger C. Cell. 2009; 139: 428-439Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Three PDE2A isoforms have been described, PDE2A1, PDE2A2, and PDE2A3, which are encoded by a single mammalian PDE2A gene and differ in their N-terminal sequences due to alternative splicing (22Rosman G.J. Martins T.J. Sonnenburg W.K. Beavo J.A. Ferguson K. Loughney K. Gene. 1997; 191: 89-95Crossref PubMed Scopus (112) Google Scholar, 23Sonnenburg W.K. Mullaney P.J. Beavo J.A. J. Biol. Chem. 1991; 266: 17655-17661Abstract Full Text PDF PubMed Google Scholar, 24Yang Q. Paskind M. Bolger G. Thompson W.J. Repaske D.R. Cutler L.S. Epstein P.M. Biochem. Biophys. Res. Commun. 1994; 205: 1850-1858Crossref PubMed Scopus (66) Google Scholar). The isoform 3 N terminus serves as a myristoylation site for membrane anchoring (34Russwurm C. Zoidl G. Koesling D. Russwurm M. J. Biol. Chem. 2009; 284: 25782-25790Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), but no function is known for the N termini of isofor" @default.
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