Selective adenosine A3 receptor stimulation inhibits voltage-dependent Ca2+ currents and Ca2+-activated K+ conductances in rat dorsal root ganglion neurons

Interest has been focused in recent years on the analgesic effects exerted by adenosine in different in vivo models of acute and chronic pain. Adenosine is an endogenous neuromodula tor that acts on four metabotropic receptors: A1 and A3 receptors coupled to adenylyl cyclase inhibition, and A2A and A2B coupled to adenylyl cyclase stimulation. Preclinical and clinical studies demonstrate that A1 receptor and its agonists exert antinociceptive effects. However, the therapeutic utility of these compounds is limited by adverse cardiovascular and central side effects. Recent preclinical observations also indicate that A3 receptors (A3R), which are known to be free fromcardiovascular side effects, exerts a powerful analgesic action in in vivo rodent models of experimental neuropathic pain, such as spinal nerve ligation or chemotherapy-induced peripheral neuropathy [1,2]. However, the cellular and molecular basis of A3R-mediated antinociception are still unknown. In this study we investigate whether the A3R agonist Cl-IB-MECAmodulates excitability in dorsal root ganglion (DRG) neurons, which are the primary sensory peripheral afference of pain. Dissociated rat DRG neurons were tested for their responsiveness to the selective A3R agonist Cl-IBMECA and to the endogenous ligand adenosine (Ado) in the absence or presence of different A3R antagonists. Patch clamp recordings from primary cultures of rat DRG neurons were performed as described [3]. Exogenous application of Cl-IB-MECA concentration dependently (0.1-100 nM) inhibited voltage-gated outward currents evoked by a ramp protocol (from +80 mV to -120 mV, 800 ms duration) in medium- and small-sized DRG neurons. The I-V relationship of Cl-IB-MECA-inhibited current, investigated by applying a voltage step protocol (13 steps from -40 to +80 mV, 200 ms, Vh = -80 mV) was consistent with the inhibition of non-inactivating and depolarization-activated K+ currents (as confirmed by Cs+-replacement experimetns). Cl-IB-MECA effect was mimicked by adenosine (Ado: 30 μM) and prevented in the presence of the selective A3 antagonists MRS1523 and VUF5574 (100 nM) but not by MRS1220 (0.1-1 μM). On the other hand, adenosine-mediated K+ current inhibition was only partially blocked either by MRS1523 or VUF5574. In the presence of 1 mM extracellular Cd2+, which inhibits Ca2+ entry from voltage-operated Ca+2 channels (VOCCs), the effect of Cl-IB-MECAwas completely prevented, thus demonstrating that the K+ conductances inhibited by the A3R agonist belong to Ca2+-activated K+ channel (KCa) family. In order to verify whether the A3R agonist directly inhibits KCa or blocks Ca2+ entry from VOCCs and, in turn, decreases outward KCa currents, we blocked all K+ channels by replacing intra- and extracellular K+ by equimolar Cs+. Under these experimental conditions, a Cd2+-sensitive inward current appeared in the ramp traces at voltages from -30 to +40mV, coherentlywith VOCCs gating. This inward Ca2+-mediated current was inhibited by 100 nM Cl-IB-MECA application. Present data demonstrate that adenosineA3R activation inhibits Ca2+ entry fromVOCCs and, in turn, decreases outwardK+ currents evoked by a voltage ramp protocol in rat DRG neurons. These effects might represent themolecular basis of A3-mediated antinociceptive effects observed in vivo, since Ca+2 current inhibition would result in hampering of nociceptive neurotransmission from DRG to CNS.