PP  Vol.4 No.1 , January 2013
Misoprostol and the Sildenafil analog (PHAR-0099048) Modulate Cellular Efflux of cAMP and cGMP Differently
Abstract: In the present study we have characterized ATP-dependent transport of cAMP and cGMP in physiological, but also supraphysiological concentrations. The uptake into inside-out vesicles from human erythrocytes could be dissected into two components with high and low affinity. The respective Km-values were 30.8 ± 5.2 and 352 ± 26 μM for cAMP and 2.6 ± 0.4 and 260 ± 15 μM for cGMP. The two cyclic nucleotides were unable to mutually inhibit cellular efflux for concentrations up to about 100 μM. At higher concentrations the inhibition curve showed a steep fall. The IC50-value for cAMP reduction of high affinity [3H]-cGMP transport was 695 ± 9 μM. The respective value for cGMP inhibition of [3H]-cAMP efflux was 284 ± 20 μM. These observations are compatible with two selective high affinity transport systems. Other endogenous substances such as prostaglandins did not discriminate between cyclic nucleotide transport. The IC50 values for inhibition of [3H]-cAMP and [3H]-cGMP were 4.1 and 4.2 μM for PGE1, 2.7 and 4.4 μM for PGE2, respectively. However, the prostaglandin analog misoprostol discriminated distinctly between cAMP and cGMP transport with respective IC50-values of 4.5 and 24 μM. The assumption that the specific PDE5-inhibitor sildenafil could distinguish between the two cyclic nucleotides was disproved with respective IC50 values of 3.8 and 2.9 μM for inhibition of [3H]-cAMP and [3H]-cGMP, respectively. However, at least one sildenafil analog (PHAR0099048) showed a clear difference with respective IC50 values of 2.0 and 0.52 μM. The other tested sildenafil analogs showed no or minor ability to discriminate with IC50 values of 0.16 and 0.17 μM for IS-39213, and 0.35 and 0.16 μM for IS-60049, respectively. In agreement with previous reports, the present study shows that proteins responsible for cyclic nucleotide transport are multiorganic anion pumps. However, the observation that drug analogs may discriminate between these two efflux systems makes them potential drug targets.
Cite this paper: E. Ørvoll, R. Lysaa, A. Ravna and G. Sager, "Misoprostol and the Sildenafil analog (PHAR-0099048) Modulate Cellular Efflux of cAMP and cGMP Differently," Pharmacology & Pharmacy, Vol. 4 No. 1, 2013, pp. 104-109. doi: 10.4236/pp.2013.41015.

[1]   G. Jedlitschky, B. Burchell and D. Keppler, “The Multidrug Resistance Protein 5 Functions as an ATP-Dependent Export Pump for Cyclic Nucleotides,” The Journal of Biological Chemistry, Vol. 275, No. 39, 2000, pp. 30069-30074. doi:10.1074/jbc.M005463200

[2]   Z. S. Chen, K. Lee and G. D. Kruh, “Transport of Cyclic Nucleotides and Estradiol 17-Beta-D-Glucuronide by Multidrug Resistance Protein 4. Resistance to 6-Mercaptopurine and 6-Thioguanine,” The Journal of Biological Chemistry, Vol. 276, No. 36, 2001, pp. 33747-33754. doi:10.1074/jbc.M104833200

[3]   M. Adachi, G. Reid and J. D. Schuetz, “Therapeutic and Biological Importance of Getting Nucleotides out of Cells: A Case for the ABC Transporters, MRP4 and 5,” Advanced Drug Delivery Reviews, Vol. 54, No. 10, 2002, pp. 1333-1342. doi:10.1016/S0169-409X(02)00166-7

[4]   P. R. Wielinga, D. H. van, I. G. Reid, J. H. Beijnen, J. Wijnholds and P. Borst, “Characterization of the MRP4- and MRP5-Mediated Transport of Cyclic Nucleotides from Intact Cells,” The Journal of Biological Chemistry, Vol. 278, No. 20, 2003, pp. 17664-17671. doi:10.1074/jbc.M212723200

[5]   Y. Guo, E. Kotova, Z. S. Chen, K. Lee, E. Hopper-Borge, M. G. Belinsky and G. D. Kruh, “MRP8 (ABCC11) Is a Cyclic Nucleotide Efflux Pump and a Resistance Factor for Fluoropyrimidines, 2’3’-Dideoxycytidine and 9’-(2’- Phosphonylmethoxyethyl)-Adenine,” The Journal of Biological Chemistry, Vol. 278, No. 32, 2003, pp. 29509-29514. doi:10.1074/jbc.M304059200

[6]   S. N. Orlov and N. V. Maksimova, “Efflux of Cyclic Adenosine Monophosphate from Cells: Mechanisms and Physiological Implications,” Biochemistry, Vol. 64, No. 2, 1999, pp. 127-135.

[7]   G. Sager, “Cyclic GMP Transporters,” Neurochemistry International, Vol. 45, No. 6, 2004, pp. 865-873. doi:10.1016/j.neuint.2004.03.017

[8]   A. Klokouzas, C. P. Wu, H. W. van Veen, M. A. Barrand and S. B. Hladky, “cGMP and Glutathione-Conjugate Transport in Human Erythrocytes,” European Journal of Biochemistry, Vol. 270, No. 18, 2003, pp. 3696-3708. doi:10.1046/j.1432-1033.2003.03753.x

[9]   C. P. Wu, H. Woodcock, S. B. Hladky and M. A. Barrand, “cGMP (Guanosine 3’,5’-Cyclic Monophosphate) Transport across Human Erythrocyte Membranes,” Biochemical Pharmacology, Vol. 69, No. 8, 2005, pp. 1257-1262. doi:10.1016/j.bcp.2005.02.005

[10]   C. J. de Wolf, H. Yamaguchi, D. H. van, I. P. R. Wielinga, S. L. Hundscheid, N. Ono, G. L. Scheffer, H. M. de, J. D. Schuetz, J. Wijnholds and P. Borst, “cGMP Transport by Vesicles from Human and Mouse Erythrocytes,” The FEBS Journal, Vol. 274, No. 2, 2007, pp. 439-450. doi:10.1111/j.1742-4658.2006.05591.x

[11]   M. Rius, J. Humel-Eisenbeiss and D. Keppler, “ATP-Dependent Transport of Leukotrienes B4 and C4 by the Multidrug Resistance Protein ABCC4 (MRP4),” The Journal of Pharmacology and Experimental Therapeutics, Vol. 324, No. 1, 2008, pp. 86-94. doi:10.1124/jpet.107.131342

[12]   E. Boadu and G. Sager, “Reconstitution of ATP-Dependent cGMP Transport into Proteoliposomes by Membrane Proteins from Human Erythrocytes,” Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 64, No. 1, 2004, pp. 41-48. doi:10.1080/00365510410003895

[13]   E. Sundkvist, R. Jaeger and G. Sager, “Pharmacological Characterization of the ATP-Dependent Low K(m) Guanosine 3’,5’-Cyclic Monophosphate (cGMP) Transporter in Human Erythrocytes,” Bi-ochemical Pharmacology, Vol. 63, No. 5, 2002, pp. 945-949. doi:10.1016/S0006-2952(01)00940-6

[14]   G. Sager, E. Orvoll, R. Lysaa, I. Kufareva, R. Abagyan and A. W. Ravna, “Novel cGMP Efflux Inhibitors— Identified by Virtual Ligand Screening (VLS) and Confirmed by Experimental Studies,” Journal of Medicinal Chemistry, Vol. 55, No. 7, 2012, pp. 3049-3057. doi:10.1021/jm2014666

[15]   M. J. Rindler, M. M. Bashor, N. Spitzer and M. H. Saier Jr., “Regulation of Adenosine 3’:5’-Monophosphate Efflux from Animal Cells,” The Journal of Biological Chemistry, Vol. 253, No. 15, 1978, pp. 5431-5436.

[16]   L. L. Brunton and S. E. Mayer, “Extrusion of Cyclic AMP from Pigeon Erythrocytes,” The Journal of Biological Chemistry, Vol. 254, No. 19, 1979, pp. 9714-9720.

[17]   T. L. Steck, R. S. Weinstein, J. H. Straus and D. F. H. Wallach, “Inside-Out Cell Membrane Vesicles: Preparation and Purification,” Science, Vol. 168, No. 928, 1970, pp. 255-257. doi:10.1126/science.168.3928.255

[18]   G. L. Ellman, K. D. Courtney, V. Andres Jr. and R. M. Featherstone, “A New and Rapid Colorometric Determination of Acetylcholinesterase Activity,” Biochemical Pharmacology, Vol. 7, 1961, pp. 88-95. doi:10.1016/0006-2952(61)90145-9

[19]   G. A. McPherson, “A Mathemathical Approach to Receptor Characterization,” In: R. E. Williams, R. A. Glennon and P. B. Timmermans, Receptor Pharmacology and Function, Marcel Dekker Inc., New York, 1989, pp. 47-84.

[20]   T. C. Chou, “Derivation and Properties of Michaelis-Menten Type and Hill Type Equations for reference Ligands,” Journal of Theoretical Biology, Vol. 39, No. 2, 1976, pp. 253-276. doi:10.1016/0022-5193(76)90169-7

[21]   C. Schultz, S. Vaskinn, H. Kildalsen and G. Sager, “Cyclic AMP Stimulates the Cyclic GMP Egression Pump in Human Erythrocytes: Effects of Probenecid, Verapamil, Progesterone, Theophylline, IBMX, Forskolin, and Cyclic AMP on Cyclic GMP Uptake and Association to Inside-Out Vesicles,” Bio-chemistry, Vol. 37, No. 4, 1998, pp. 1161-1166. doi:10.1021/bi9713409

[22]   P. Borst, C. de Wolf and K. van de Wetering, “Multidrug Resistance-Associated Proteins 3, 4, and 5,” Pflugers Archiv: European Journal of Physiology, Vol. 453, No. 5, 2007, pp. 661-673.

[23]   J. D. Corbin, S. H. Francis and D. J. Webb, “Phosphodiesterase Type 5 as a Pharmacologic Target in Erectile Dysfunction,” Urology, Vol. 60, No. 2, 2002, pp. 4-11. doi:10.1016/S0090-4295(02)01686-2

[24]   G. D. Holman, “Cyclic AMP Transport in Human Erythrocyte Ghosts,” Biochimica et Biophysica Acta, Vol. 508, No. 1, 1978, pp. 174-183. doi:10.1016/0005-2736(78)90199-2

[25]   G. Sager, A. Orbo, R. H. Pettersen and K. E. Kj?rstad, “Export of Guanosine 3’,5’-Cyclic Monophosphate (cGMP) from Human Erythrocytes Characterized by Inside-Out Membrane Vesicles,” Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 56, No. 4, 1996, pp. 289-293. doi:10.3109/00365519609090579

[26]   E. Boadu, S. Vaskinn, E. Sundkvist, R. Jaeger and G. Sager, “Inhibition by Guanosine Cyclic Monophosphate (cGMP) Analogues of Uptake of [3H]3’,5’-cGMP without Stimulation of ATPase Activity in Human Erythrocyte Inside-Out Vesicles,” Biochemical Phar-macology, Vol. 62, No. 4, 2001, pp. 425-429. doi:10.1016/S0006-2952(01)00682-7

[27]   R. A. van Aubel, P. H. Smeets, J. J. Van Den Heuvel and F. G. Russel, “Human Organic Anion Transporter MRP4 (ABCC4) Is an Efflux Pump for the Purine End Metabolite Urate with Multiple Allosteric Substrate Binding Sites,” American Journal of Physiology. Renal Physiology, Vol. 288, No. 2, 2005, pp. F327-F333. doi:10.1152/ajprenal.00133.2004

[28]   H. G. Wittgen, J. J. Van Den Heuvel, E. Krieger, G. Schaftenaar, F. G. Russel and J. B. Koenderink, “Phenylalanine 368 of Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Plays a Crucial Role in Substrate-Specific Transport Activity,” Biochemical Pharmacology, Vol. 84, No. 3, 2012, pp. 366-373. doi:10.1016/j.bcp.2012.04.012

[29]   L. Lai and T. M. Tan, “Role of Glutathione in the Multidrug Resistance Protein 4 (MRP4/ABCC4)-Mediated Efflux of cAMP and Resistance to Purine Analogues,” Biochemical Journal, Vol. 361, No. 3, 2002, pp. 497-503. doi:10.1042/0264-6021:3610497

[30]   Z. P. Lin, Y. L. Zhu, D. R. Johnson, K. P. Rice, T. Nottoli, B. C. Hains, J. McGrath, S. G. Waxman and A. C. Sartorelli, “Disruption of cAMP and PGE2 Transport by Mrp4 Deficiency Alters cAMP-Mediated Signaling and Nociceptive Response,” Molecular Pharmacology, Vol. 73, No. 1, 2008, pp. 243-251. doi:10.1124/mol.107.039594

[31]   Y. Sassi, L. Lipskaia, G. Vandecasteele, V. O. Nikolaev, S. N. Hatem, A. F. Cohen, F. G. Russel, N. Mougenot, C. Vrignaud, P. Lechat, A. M. Lompre and J. S. Hulot, “Multidrug Resistance-Associated Protein 4 Regulates cAMP-Dependent Signaling Pathways and Controls Human and Rat SMC Proliferation,” Journal of Clinical Investigation, Vol. 118, No. 8, 2008, pp. 2747-2757. doi:10.1172/JCI35067

[32]   S. Copsel, C. Garcia, F. Diez, M. Vermeulem, A. Baldi, L. G. Bianciotti, F. G. Russel, C. Shayo and C. Davio, “Multidrug Resistance Protein 4 (MRP4/ ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation,” The Journal of Biological Chemistry, Vol. 286, No. 9, 2011, pp. 6979-6988. doi:10.1074/jbc.M110.166868

[33]   M. R. Rodriguez, F. Diez, M. S. Ventimiglia, V. Morales, S. Copsel, M. S. Vatta, C. A. Davio and L. G. Bianciotti, “Atrial Natriuretic Factor Stimulates Efflux of cAMP in Rat Exocrine Pancreas via Multidrug Resistance-Associated Proteins,” Gastroenterology, Vol. 140, No. 4, 2011, pp. 1292-1302. doi:10.1053/j.gastro.2010.12.053

[34]   G. Jedlitschky, K. Tirschmann, L. E. Lubenow, H. K. Nieuwenhuis, J. W. Akkerman, A. Greinacher and H. K. Kroemer, “The Nucleotide Transporter MRP4 (ABCC4) Is Highly Expressed in Human Platelets and Present in Dense Granules, Indicating a Role in Mediator Storage,” Blood, Vol. 104, No. 12, 2004, pp. 3603-3610. doi:10.1182/blood-2003-12-4330

[35]   X. B. Wu, B. Brune, F. von Appen and V. Ullrich, “Efflux of Cyclic GMP from Activated Human Platelets,” Molecular Pharmacology, Vol. 43, No. 4, 1993, pp. 564-568.

[36]   W. Radziszewski, M. Chopra, A. Zembowicz, R. Gryglewski, L. J. Ignarro and G. Chaudhuri, “Nitric Oxide Donors Induce Extrusion of Cyclic GMP from Isolated Human Blood Platelets by a Mechanism Which May Be Modulated by Prostaglandins,” International Journal of Cardiology, Vol. 51, No. 3, 1995, pp. 211-220. doi:10.1016/0167-5273(95)02427-X

[37]   Z. E. Sauna, K. Nandigama and S. V. Ambudkar, “Multidrug Resistance Protein 4 (ABCC4)-Mediated ATP Hydrolysis: Effect of Transport Substrates and Characterization of the Post-Hydrolysis Transition State,” The Journal of Biological Chemistry, Vol. 279, No. 47, 2004, pp. 48855-48864. doi:10.1074/jbc.M408849200

[38]   S. A. Andric, T. S. Kostic and S. S. Stojilkovic, “Contribution of Multidrug Resistance Protein MRP5 in Control of cGMP Intracellular Signaling in Anterior Pituitary Cells,” Endocrinology, Vol. 147, No. 7, 2006, pp. 3435-3445. doi:10.1210/en.2006-0091

[39]   G. Reid, P. Wielinga, N. Zelcer, D. H. van, I, A. Kuil, M. de Haas, J. Wijnholds and P. Borst, “The Human Multidrug Resistance Protein MRP4 Functions as a Prostaglandin Efflux Transporter and Is Inhibited by Nonsteroidal Antiinflammatory Drugs,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, No. 16, 2003, pp. 9244-9249. doi:10.1073/pnas.1033060100