Modulation of the dinucleotide receptor present in rat midbrain synaptosomes by adenosine and ATP

Diadenosine polyphosphates activate dinucleotide receptors in rat midbrain synaptic terminals. The agonist with highest affinity at this receptor, diadenosine pentaphosphate (Ap5A), elicits Ca2+ transients at concentrations ranging from 10−7 to 10−3 M with a single-phase curve and an EC50 value of 56.21±1.82 μM. Treatment of synaptosomal preparations with alkaline phosphatase (AP) changes the dose-response control curve into a biphasic one presenting two EC50 values of 6.47±1.25 nM and 11.16±0.83 μM respectively. The adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) reversed the biphasic concentration-response for Ap5A curve in the presence of AP, to a monophasic one with an EC50 value of 76.05±7.51 μM. The application of adenosine deaminase produced the same effect as DPCPX, the EC50 value for Ap5A, in the presence of AP being 18.62±4.03 μM. Activation of the adenosine A1 receptor by means of cyclohexyladenosine (CHA) shifted the dose response curve for Ap5A to the left, resulting in a monophasic curve with an EC50 of 5.01±0.02 pM. The destruction of extrasynaptosomal nucleotides by AP or the addition of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a broad P2 antagonist compound, enhance maximal effect of the Ap5A up to 55.6% on the dose response curve, thus suggesting a negative modulation by P2 receptors. In a summary, ATP and adenosine present at the extra-synaptosomal space, are relevant natural modulators of the dinucleotide receptor, via P2 and adenosine A1 receptors respectively. Keywords: ATP, diadenosine polyphosphates, adenosine, dinucleotide receptor, alkaline phosphatase, adenosine receptor Introduction Diadenosine polyphosphates, also termed adenine dinucleotides, are a family of nucleotidic compounds formed by two adenosines linked by a variable number of phosphates (Baxi & Vishwanatha, 1995; McLennan, 1992). Diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A), and ATP, are present in rat brain synaptosomes, and are stored in secretory vesicles by means of a high affinity nucleotide vesicular transporter (Gualix et al., 1997). ATP and adenine dinucleotides are released after synaptic terminal stimulation (Potter & White, 1980; Pintor et al., 1992) and activate different purinergic receptors in neural and non-neural cells once in the extracellular medium (Hoyle, 1990; Pintor et al., 1997b). Actions of diadenosine polyphosphates mediated by purine receptors in the central nervous system are the modulation of the firing rate in cortical neurones (Stone & Perkins, 1981), inhibition of the synaptic transmission in hippocampal slices (Klishin et al., 1994) and the facilitation of action potentials in locus coeruleus neurones (Frohlich et al., 1996). In rat, guinea-pig and deermouse synaptosomes the presence of independent receptors for adenine dinucleotides and ATP have been reported (Pintor & Miras-Portugal, 1995b; Pintor et al., 1997b; Pivorun & Nordone, 1996). Considering that all of these are receptor operated Ca2+ channels, they might be involved in facilitatory presynaptic mechanisms in central neurones (Pintor & Miras-Portugal, 1995b; Pintor et al., 1997b). The Ca2+ signal mediated by these presynaptic receptors can be modulated by the action of protein kinases and phosphatases. Effectors inducing the activation of either protein kinase A (PKA) and protein kinase C (PKC) result in a considerable Ca2+ signal reduction. (Pintor et al., 1997a). ATP and its breakdown product adenosine can activate P2 and adenosine receptors, respectively, promoting modulatory mechanisms involved, in many cases, in the presynaptic control of transmitter release (Schubert et al., 1995; Ribeiro, 1995). Keeping in view the fact that diadenosine polyphosphates, ATP and adenosine can co-exist extracellularly in neural models and in synaptosomal preparations, and given their physiological importance, we sought to describe their effect of the Ca2+ responses elicited by Ap5A through the dinucleotide receptor.