Background We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs). Methodology/Principal Findings The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3 = PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β1- and β2-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β1- and β2-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation. Conclusion/Significance Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.
References
[1]
Koyama H, Bornfeldt KE, Fukumoto S, Nishizawa Y (2001) Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation. J Cell Physiol 186: 1–10.
[2]
McDaniel NL, Rembold CM, Murphy RA (1994) Cyclic nucleotide dependent relaxation in vascular smooth muscle. Can J Physiol Pharmacol 72: 1380–1385.
[3]
Omori K, Kotera J (2007) Overview of PDEs and their regulation. Circ Res 100: 309–327.
[4]
Maurice DH, Palmer D, Tilley DG, Dunkerley HA, Netherton SJ, et al. (2003) Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol 64: 533–546.
[5]
Phillips PG, Long L, Wilkins MR, Morrell NW (2005) cAMP phosphodiesterase inhibitors potentiate effects of prostacyclin analogs in hypoxic pulmonary vascular remodeling. Am J Physiol Lung Cell Mol Physiol 288: L103–L115.
[6]
Fischmeister R, Castro LR, Abi-Gerges A, Rochais F, Jurevicius J, et al. (2006) Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res 99: 816–828.
[7]
Xiang YK (2011) Compartmentalization of β-adrenergic signals in cardiomyocytes. Circ Res 109: 231–244.
[8]
Mika D, Leroy J, Vandecasteele G, Fischmeister R (2012) PDEs create local domains of cAMP signaling. J Mol Cell Cardiol 52: 323–329.
[9]
Castro LR, Verde I, Cooper DM, Fischmeister R (2006) Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. Circulation 113: 2221–2228.
[10]
Delpy E, Coste H, Gouville AC (1996) Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3. Br J Pharmacol 119: 471–478.
[11]
Piggott LA, Hassell KA, Berkova Z, Morris AP, Silberbach M, et al. (2006) Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments. J Gen Physiol 128: 3–14.
[12]
Nausch LW, Ledoux J, Bonev AD, Nelson MT, Dostmann WR (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci U S A 105: 365–370.
[13]
Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279: 37215–37218.
[14]
Nikolaev VO, Lohse MJ (2006) Monitoring of cAMP synthesis and degradation in living cells. Physiology (Bethesda) 21: 86–92.
[15]
Thyberg J, Hedin U, Sjolund M, Palmberg L, Bottger BA (1990) Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 10: 966–990.
[16]
Dunkerley HA, Tilley DG, Palmer D, Liu H, Jimmo SL, et al. (2002) Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues. Mol Pharmacol 61: 1033–1040.
[17]
Rybalkin SD, Bornfeldt KE, Sonnenburg WK, Rybalkina IG, Kwak KS, et al. (1997) Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype. J Clin Invest 100: 2611–2621.
[18]
Liu H, Palmer D, Jimmo SL, Tilley DG, Dunkerley HA, et al. (2000) Expression of phosphodiesterase 4D (PDE4D) is regulated by both the cyclic AMP-dependent protein kinase and mitogen-activated protein kinase signaling pathways. A potential mechanism allowing for the coordinated regulation of PDE4D activity and expression in cells. J Biol Chem 275: 26615–26624.
[19]
Vallot O, Combettes L, Jourdon P, Inamo J, Marty I, et al. (2000) Intracellular Ca2+ handling in vascular smooth muscle cells is affected by proliferation. Arterioscler Thromb Vasc Biol 20: 1225–1235.
[20]
Thompson WJ, Appleman MM (1971) Characterization of cyclic nucleotide phosphodiesterases of rat tissues. J Biol Chem 246: 3145–3150.
[21]
Saucerman JJ, McCulloch AD (2006) Cardiac β-adrenergic signaling: from subcellular microdomains to heart failure. Ann N Y Acad Sci 1080: 348–361.
[22]
Wachten S, Masada N, Ayling LJ, Ciruela A, Nikolaev VO, et al. (2010) Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells. J Cell Sci 123: 95–106.
[23]
Boess FG, Hendrix M, van der Staay FJ, Erb C, Schreiber R, et al. (2004) Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance. Neuropharmacology 47: 1081–1092.
[24]
Sudo T, Tachibana K, Toga K, Tochizawa S, Inoue Y, et al. (2000) Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity. Biochem Pharmacol 59: 347–356.
[25]
Rich TC, Tse TE, Rohan JG, Schaack J, Karpen JW (2001) In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J Gen Physiol 118: 63–78.
[26]
Wells JN, Miller JR (1988) Methylxanthine inhibitors of phosphodiesterases. Methods Enzymol 159: 489–496.
[27]
Goncalves RL, Lugnier C, Keravis T, Lopes MJ, Fantini FA, et al. (2009) The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue. Eur J Pharmacol 620: 78–83.
[28]
Smith SJ, Cieslinski LB, Newton R, Donnelly LE, Fenwick PS, et al. (2004) Discovery of BRL 50481. Mol Pharmacol 66: 1679–1689.
[29]
Leblais V, Delannoy E, Fresquet F, Begueret H, Bellance N, et al. (2008) β-Adrenergic relaxation in pulmonary arteries: preservation of the endothelial nitric oxide-dependent β2 component in pulmonary hypertension. Cardiovasc Res 77: 202–210.
[30]
Hoffmann C, Leitz MR, Oberdorf-Maass S, Lohse MJ, Klotz KN (2004) Comparative pharmacology of human β-adrenergic receptor subtypes characterization of stably transfected receptors in CHO cells. Naunyn Schmiedebergs Arch Pharmacol 369: 151–159.
[31]
Palmer D, Maurice DH (2000) Dual expression and differential regulation of phosphodiesterase 3A and phosphodiesterase 3B in human vascular smooth muscle: implications for phosphodiesterase 3 inhibition in human cardiovascular tissues. Mol Pharmacol 58: 247–252.
[32]
Orallo F, Alvarez E, Basaran H, Lugnier C (2004) Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin. Naunyn Schmiedebergs Arch Pharmacol 370: 452–463.
[33]
Rose RJ, Liu H, Palmer D, Maurice DH (1997) Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity. Br J Pharmacol 122: 233–240.
[34]
Leroy J, Abi-Gerges A, Nikolaev VO, Richter W, Lechene P, et al. (2008) Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases. Circ Res 102: 1091–1100.
[35]
Sassi Y, Lipskaia L, Vandecasteele G, Nikolaev VO, Hatem SN, et al. (2008) Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation. J Clin Invest 118: 2747–2757.
[36]
von Hayn K, Werthmann RC, Nikolaev VO, Hommers LG, Lohse MJ, et al. (2010) Gq-mediated Ca2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells. Am J Physiol Cell Physiol 298: C324–C332.
[37]
Trochu JN, Leblais V, Rautureau Y, Beverelli F, Le Marec H, et al. (1999) β3-Adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium-derived nitric oxide in rat thoracic aorta. Br J Pharmacol 128: 69–76.
[38]
Brawley L, Shaw AM, MacDonald A (2000) β1-, β2- and atypical β-adrenoceptor-mediated relaxation in rat isolated aorta. Br J Pharmacol 129: 637–644.
[39]
Guimaraes S, Moura D (2001) Vascular adrenoceptors: an update. Pharmacol Rev 53: 319–356.
[40]
Ostrom RS, Liu X, Head BP, Gregorian C, Seasholtz TM, et al. (2002) Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains. Mol Pharmacol 62: 983–992.
[41]
Rautureau Y, Toumaniantz G, Serpillon S, Jourdon P, Trochu JN, et al. (2002) β3-adrenoceptor in rat aorta: molecular and biochemical characterization and signalling pathway. Br J Pharmacol 137: 153–161.
[42]
Nikolaev VO, Bunemann M, Schmitteckert E, Lohse MJ, Engelhardt S (2006) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling. Circ Res 99: 1084–1091.
[43]
Richter W, Day P, Agrawal R, Bruss MD, Granier S, et al. (2008) Signaling from β1- and β2-adrenergic receptors is defined by differential interactions with PDE4. EMBO J 27: 384–393.
[44]
Degerman E, Belfrage P, Manganiello VC (1997) Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem 272: 6823–6826.
[45]
Rovner AS, Murphy RA, Owens GK (1986) Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells. J Biol Chem 261: 14740–14745.
[46]
Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801.
