Hide and seek: a comparative autoradiographic in vitro investigation of the adenosine A3 receptor

PurposeSince the adenosine A3 receptor (A3R) is considered to be of high clinical importance in the diagnosis and treatment of ischaemic conditions (heart and brain), glaucoma, asthma, arthritis, cancer and inflammation, a suitable and selective A3R PET tracer such as [18F]FE@SUPPY would be of high clinical value for clinicians as well as patients. A3R was discovered in the late 1990s, but there is still little known regarding its distribution in the CNS and periphery. Hence, in autoradiographic experiments the distribution of A3R in human brain and rat tissues was investigated and the specific binding of the A3R antagonist FE@SUPPY and MRS1523 compared. Immunohistochemical staining (IHC) experiments were also performed to validate the autoradiographic findings.MethodsFor autoradiographic competition experiments human post-mortem brain and rat tissues were incubated with [125I]AB-MECA and highly selective compounds to block the other adenosine receptor subtypes. Additionally, IHC was performed with an A3 antibody.ResultsSpecific A3R binding of MRS1523 and FE@SUPPY was found in all rat peripheral tissues examined with the highest amounts in the spleen (44.0 % and 46.4 %), lung (44.5 % and 45.0 %), heart (39.9 % and 42.9 %) and testes (27.4 % and 29.5 %, respectively). Low amounts of A3R were found in rat brain tissues (5.9 % and 5.6 %, respectively) and human brain tissues (thalamus 8.0 % and 9.1 %, putamen 7.8 % and 8.2 %, cerebellum 6.0 % and 7.8 %, hippocampus 5.7 % and 5.6 %, caudate nucleus 4.9 % and 6.4 %, cortex 4.9 % and 6.3 %, respectively). The outcome of the A3 antibody staining experiments complemented the results of the autoradiographic experiments.ConclusionThe presence of A3R protein was verified in central and peripheral tissues by autoradiography and IHC. The specificity and selectivity of FE@SUPPY was confirmed by direct comparison with MRS1523, providing further evidence that [18F]FE@SUPPY may be a suitable A3 PET tracer for use in humans.

[1]  A. Pérez-Samartín,et al.  A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve , 2014, Glia.

[2]  K. Varani,et al.  A3 Adenosine Receptors in Human Neutrophils and Promyelocytic HL60 Cells: A Pharmacological and Biochemical Study , 2002 .

[3]  C. Monitto,et al.  Localization of the A3 adenosine receptor gene (ADORA3) to human chromosome 1p. , 1995, Genomics.

[4]  Mingzhang Gao,et al.  Simple synthesis of new carbon-11-labeled 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives for PET imaging of A₃ adenosine receptor. , 2014, Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine.

[5]  P. Toselli,et al.  Activity of the A3 adenosine receptor gene promoter in transgenic mice: characterization of previously unidentified sites of expression , 2002, FEBS letters.

[6]  C. Psarropoulou,et al.  The A3 adenosine receptor agonist 2-Cl-IB-MECA facilitates epileptiform discharges in the CA3 area of immature rat hippocampal slices , 2004, Epilepsy Research.

[7]  R. Lin,et al.  Stimulation of Adenosine A3 Receptors in Cerebral Ischemia: Neuronal Death, Recovery, or Both? , 1999, Annals of the New York Academy of Sciences.

[8]  T. Dunwiddie,et al.  Activation of Hippocampal Adenosine A3 Receptors Produces a Desensitization of A1 Receptor-Mediated Responses in Rat Hippocampus , 1997, The Journal of Neuroscience.

[9]  R. Gambari,et al.  Elevated Expression of A3 Adenosine Receptors in Human Colorectal Cancer Is Reflected in Peripheral Blood Cells , 2004, Clinical Cancer Research.

[10]  P. Fishman,et al.  Targeting the A3 adenosine receptor for cancer therapy: inhibition of prostate carcinoma cell growth by A3AR agonist. , 2003, Anticancer research.

[11]  A. Mendonça,et al.  Adenosine receptors in the nervous system: pathophysiological implications , 2002, Progress in Neurobiology.

[12]  K. Klotz,et al.  Adenosine receptors and cancer. , 2011, Handbook of experimental pharmacology.

[13]  E. Benarroch.  Adenosine and its receptors , 2008, Neurology.

[14]  F. Fazio,et al.  Design, radiosynthesis, and biodistribution of a new potent and selective ligand for in vivo imaging of the adenosine A(2A) receptor system using positron emission tomography. , 2000, Journal of medicinal chemistry.

[15]  R. Cunha,et al.  Adenosine A3 receptors are located in neurons of the rat hippocampus , 2003, Neuroreport.

[16]  Kyong-Tai Kim,et al.  Pharmacological characterization of adenosine receptors in PGT‐β mouse pineal gland tumour cells , 2001 .

[17]  K. Jacobson,et al.  Synthesis and characterization of [76Br]-labeled high-affinity A3 adenosine receptor ligands for positron emission tomography. , 2009, Nuclear medicine and biology.

[18]  C. Simioni,et al.  Adenosine receptors and cancer. , 2011, Biochimica et biophysica acta.

[19]  A. Gubitz,et al.  Tissue distribution of adenosine receptor mRNAs in the rat , 1996, British journal of pharmacology.

[20]  K. Varani,et al.  The A3 adenosine receptor: an enigmatic player in cell biology. , 2008, Pharmacology & therapeutics.

[21]  L. Nics,et al.  [18F]FE@SUPPY and [18F]FE@SUPPY:2--metabolic considerations. , 2010, Nuclear medicine and biology.

[22]  Wyeth Wasserman,et al.  Structure and function of adenosine receptors and their genes , 2000, Naunyn-Schmiedeberg's Archives of Pharmacology.

[23]  Gil Ohana,et al.  The A3 Adenosine Receptor Is Highly Expressed in Tumor versus Normal Cells , 2004, Clinical Cancer Research.

[24]  P. Fishman,et al.  A3 adenosine receptor as a target for cancer therapy. , 2002, Anti-cancer drugs.

[25]  L. Berrino,et al.  The role of A3 adenosine receptors in central regulation of arterial blood pressure , 1998, British journal of pharmacology.

[26]  K. Varani,et al.  Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line , 2001, British journal of pharmacology.

[27]  K. Jacobson,et al.  Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at human A(2B) adenosine receptors. , 2000, Journal of medicinal chemistry.

[28]  P. Fishman,et al.  Agonists and Antagonists: Molecular Mechanisms and Therapeutic Applications , 2010 .

[29]  Identification of adenosine A1 and A3 receptor subtypes in rat pial and intracerebral arteries , 2004, Neuroscience Letters.

[30]  L. Nics,et al.  Automatisation and First Evaluation of [18F]FE@SUPPY:2, an AlternativePET-Tracer for the Adenosine A3 Receptor: A Comparison with[18F]FE@SUPPY , 2009 .

[31]  W. Wadsak,et al.  Radiosynthesis of the adenosine A3 receptor ligand 5-(2-[18F]fluoroethyl) 2,4-diethyl-3-(ethylsulfanylcarbonyl)- 6-phenylpyridine-5-carboxylate ([18F]FE@SUPPY) , 2008 .

[32]  Richard A Stone,et al.  Knockout of A3 adenosine receptors reduces mouse intraocular pressure. , 2002, Investigative ophthalmology & visual science.

[33]  K. Jacobson,et al.  A physiological role of the adenosine A3 receptor: sustained cardioprotection. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Ferrari,et al.  Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells , 2001, British journal of pharmacology.

[35]  S. Moro,et al.  Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists. , 1999, Journal of medicinal chemistry.

[36]  D. Weaver,et al.  [125I]4-Aminobenzyl-5′-N-methylcarboxamidoadenosine ([125I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain , 1997, Brain Research.

[37]  S. Rivkees,et al.  Are A3 adenosine receptors expressed in the brain? , 2000, Neuroreport.

[38]  James A. Bristol,et al.  Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes , 2004, Naunyn-Schmiedeberg's Archives of Pharmacology.

[39]  P. Conn,et al.  Protein Kinase C and A3 Adenosine Receptor Activation Inhibit Presynaptic Metabotropic Glutamate Receptor (mGluR) Function and Uncouple mGluRs from GTP-Binding Proteins , 1998, The Journal of Neuroscience.

[40]  R. Lin,et al.  Right Thing at a Wrong Time?. Adenosine A3 Receptors and Cerebroprotection in Stroke , 2001, Annals of the New York Academy of Sciences.

[41]  Jianjing Xue,et al.  Differential gene expression of adenosine A1, A2a, A2b, and A3 receptors in the human enteric nervous system , 2001, The Journal of comparative neurology.

[42]  W. Wadsak,et al.  Preparation and first evaluation of [(18)F]FE@SUPPY: a new PET tracer for the adenosine A(3) receptor. , 2008, Nuclear medicine and biology.

[43]  W. Wadsak,et al.  Synthesis of in vivo Metabolites of the New Adenosine A3 Receptor PET-Radiotracer [18F]FE@SUPPY , 2008 .

[44]  K. Jacobson,et al.  125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine, a high affinity radioligand for the rat A3 adenosine receptor. , 1994, Molecular pharmacology.

[45]  Kenneth A Jacobson,et al.  Recent developments in adenosine receptor ligands and their potential as novel drugs. , 2011, Biochimica et biophysica acta.

[46]  L. Nics,et al.  Radiosynthesis of a novel potential adenosine A3 receptor ligand, 5-ethyl 2,4-diethyl-3-((2-[18F]fluoroethyl)sulfanylcarbonyl)-6-phenylpyridine-5-carboxylate ([18F]FE@SUPPY:2) , 2009 .

[47]  K. Ravid,et al.  An A3-subtype adenosine receptor is highly expressed in rat vascular smooth muscle cells: its role in attenuating adenosine-induced increase in cAMP. , 1997, Microvascular research.