Alteration of A 3 adenosine receptors in human neutrophils and low frequency electromagnetic fields Katia Varani a , Stefania Gessi a , Stefania Merighi a , Valeria Iannotta a , Elena Cattabriga a , Cecilia Pancaldi a , Ruggero Cadossi b , Pier Andrea Borea a,* a Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, via Fossato di Mortara 17-19, Ferrara 44100, Italy b Laboratory of Biophysics, IGEA, Carpi, Italy Received 7 March 2003; accepted 6 June 2003 Abstract The present study was designed to evaluate the binding and functional characterization of A 3 adenosine receptors in human neutrophils exposed to low frequency, low energy, pulsing electromagnetic fields (PEMFs). Great interest has grown concerning the use of PEMF in the clinical practice for therapeutic purposes strictly correlated with inflammatory conditions. Saturation experiments performed using the high affinity and selective A 3 adenosine antagonist 5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4- triazolo[1,5-c]pyrimidine ([ 3 H]-MRE 3008F20) revealed a single class of binding sites with similar affinity in control and in PEMF treated human neutrophils (K D ¼ 2:36  0:16 and 2:45  0:15 nM, respectively). PEMFs treatment revealed that the receptor density was statistically increased (P < 0:01) (B max ¼ 451  18 and 736  25 fmol mg 1 protein, respectively). Thermodynamic data indicated that [ 3 H]-MRE 3008F20 binding in control and in PEMF-treated human neutrophils was entropy and enthalpy driven. Competition of radioligand binding by the high affinity A 3 receptor agonists, N 6 -(3-iodo-benzyl)-2-chloro-adenosine-5 0 -N-methyluronamide (Cl-IB- MECA) and N 6 -(3-iodo-benzyl)adenosine-5 0 -N-methyl-uronamide (IB-MECA), in the absence of PEMFs revealed high and low affinity values similar to those found in the presence of PEMFs. In both experimental conditions, the addition of GTP 100 mM shifted the competition binding curves of the agonists from a biphasic to a monophasic shape. In functional assays Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and their potencies were statistically increased after exposure to PEMFs. These results indicate in human neutrophils treated with PEMFs the presence of significant alterations in the A 3 adenosine receptor density and functionality. # 2003 Elsevier Inc. All rights reserved. Keywords: Adenosine A 3 receptors; Human neutrophils; [ 3 H]-MRE 3008F20 binding; Binding thermodynamics; Cyclic AMP; Pulsing electromagnetic fields (PEMFs) 1. Introduction Adenosine mediates a number of physiological func- tions through the interaction with four cell surface subtypes classified as A 1 ,A 2A ,A 2B and A 3 receptors which are coupled to G proteins [1]. The A 1 and A 3 subtypes, via Gi and Go family, mediate inhibition of the adenylyl cyclase activity, in contrast both A 2A and A 2B subtypes, coupled to Gs, determine the stimulation of cAMP production [2,3]. In particular, the A 3 adenosine receptor subtype causes ade- nylyl cyclase inhibition and phospholipase C activation [4]. The A 3 receptor plays a role in modulation of cerebral ischemia [5] with dual and opposite neuroprotective and neurodegenerative effects [6]. At cardiovascular level, A 3 adenosine receptors are involved in the ischemic heart preconditioning [7], hypotension [8] and exert many car- dioprotective effects through modulation of tissue factor release [9]. More recently, several papers point to the A 3 adenosine receptor as a promising therapeutic target on cell growth [10] and on apoptosis [11]. Adenosine, through the A 3 receptor, induces a differential effect on tumor and Biochemical Pharmacology 66 (2003) 1897–1906 0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-2952(03)00454-4 * Corresponding author. Tel.: þ39-0532-291214; fax: þ39-0532-291205. E-mail address: bpa@unife.it (P.A. Borea). Abbreviations: cAMP, cyclic AMP; [ 3 H]-MRE 3008F20, 5N-(4- methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3- e]1,2,4-triazolo[1,5-c]pyrimidine; Cl-IB-MECA, N 6 -(3-iodo-benzyl)-2- chloro-adenosine-5 0 -N-methyluronamide; IB-MECA, N 6 -(3-iodo-benzyl)a- denosine-5 0 -N-methyluronamide; Ro 20-1724, 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidinone; PEMFs, pulsing electromagnetic fields.