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

Abstract

Diadenosine polyphosphates activate dinucleotide receptors in rat midbrain synaptic terminals. The agonist with highest affinity at this receptor, diadenosine pentaphosphate (Ap(5)A), elicits Ca(2+) transients at concentrations ranging from 10(-7) to 10(-3) M with a single-phase curve and an EC(50) value of 56.21+/-1.82 microM. Treatment of synaptosomal preparations with alkaline phosphatase (AP) changes the dose-response control curve into a biphasic one presenting two EC(50) values of 6.47+/-1.25 nM and 11.16+/-0.83 microM respectively. The adenosine A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) reversed the biphasic concentration-response for Ap(5)A curve in the presence of AP, to a monophasic one with an EC(50) value of 76.05+/-7.51 microM. The application of adenosine deaminase produced the same effect as DPCPX, the EC(50) value for Ap(5)A, in the presence of AP being 18.62+/-4.03 microM. Activation of the adenosine A(1) receptor by means of cyclohexyladenosine (CHA) shifted the dose response curve for Ap(5)A to the left, resulting in a monophasic curve with an EC(50) 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 Ap(5)A 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 A(1) receptors respectively.

Figure 1

Concentration-response curve for diadenosine pentaphosphate (Ap₅A) in the presence and in the absence of alkaline phoshatase (AP). Effect of PPADS on Ap₅A response. (A) Representative experiment of the effect Ap₅A (arrows) in absence (upper traces) and in the presence (lower traces) of AP. (B) Ap₅A in concentration ranging from 10⁻⁷ to 10⁻³ M was assayed alone with 1 mg of synaptosomes as described in the Methods section. When the same protocol was carried out in the presence of alkaline phosphatase (1 u ml⁻¹) or CHA 1 μM, concentrations of Ap₅A ranging from 10⁻¹¹ to 10⁻³ M were sufficient to complete the dose response curve. 100% of effect corresponds to the maximal Ca²⁺ transient elicited by Ap₅A in the absence of any substance or enzyme. (C) Dose-response curve for Ap₅A in the presence of the P2 antagonist PPADS (50 μM) or β,γ-meATP (50 μM) preincubated for 2 min as described in the Methods section. 100% of effect corresponds to the maximal Ca²⁺ transient elicited by Ap₅A in the absence of any substance or enzyme (which corresponds to 30.53±1.64 nM). Values are the mean±s.e.mean of five experiments performed in duplicate.

Figure 2

Concentration-response curves for Ap₅A in the presence of NBTI and adenosine deaminase. The effect of the adenosine present due to the action of alkaline phosphatase (AP) was studied in two ways: (1) by blocking the adenosine transporter by means of nitrobenzyl thioinosine (NBTI) 10 μM, and (2) by adenosine destruction with adenosine deaminase 0.2 u ml⁻¹, following the protocol described in the Methods section. 100% of effect corresponds to the maximal Ca²⁺ transient elicited by Ap₅A in the absence of any substance or enzyme (which corresponds with 30.53±1.64 nM). To allow comparison, the dose-response curve for Ap₅A in the presence of alkaline phosphatase, from Figure 1, is represented as a dashed line. Values are the mean±s.e.mean of five experiments performed in duplicate.

Figure 3

Dose-response curves for diadenosine pentaphosphate in the presence of adenosine A₁ receptor modulators. The effect of the adenosine present due to the action of alkaline phosphatase (AP) was studied by blocking the adenosine A₁ receptors with antagonist DPCPX 50 nM. Also the involvement of the A₁ receptor was studied by incubating the synaptosomes with adenosine deaminase and the adenosine A₁ agonist cyclohexyladenosine (CHA) 1 μM. The effect of the protein kinase A (PKA) activator dibutyril cyclic AMP 100 μM was assayed following the protocol described in the Methods. 100% of effect corresponds to the maximal Ca²⁺ transient elicited by Ap₅A in the absence of any substance or enzyme, which value is 30.52±1.64 nM. To allow comparison, the dose-response curve for Ap₅A in the presence of alkaline phosphatase, from Figure 1, is represented as a dashed line. Values are the mean±s.e. mean of five experiments performed in duplicate.