Presence of diadenosine polyphosphates in microdialysis samples from rat cerebellum in vivo: effect of mild hyperammonemia on their receptors

Abstract

Diadenosine triphosphate (Ap(3)A), diadenosine tetraphosphate (Ap(4)A), and diadenosine pentaphosphate (Ap(5)A) have been identified in microdialysis samples from the cerebellum of conscious freely moving rats, under basal conditions, by means of a high-performance liquid chromatography method. The occurrence of Ap(3)A in the cerebellar microdyalisates is noteworthy, as the presence of this compound in the interstitial medium in neural tissues has not been previously described. The concentrations measured for the diadenosine polyphosphates in the cerebellar dialysate were (in nanomolar) 10.5 ± 2.9, 5.4 ± 1.2, and 5.8 ± 1.3 for Ap(3)A, Ap(4)A, and Ap(5)A, respectively. These concentrations are in the range that allows the activation of the presynaptic dinucleotide receptor in nerve terminals. However, a possible interaction of these dinucleotides with other purinergic receptors cannot be ruled out, as rat cerebellum expresses a variety of P2X or P2Y receptors susceptible to be activated by diadenosine polyphosphates, such as the P2X1-4, P2Y(1), P2Y(2), P2Y(4), and P2Y(12) receptors, as demonstrated by quantitative real-time PCR. Also, the ecto-nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP3, able to hydrolyze the diadenosine polyphosphates and terminate their extracellular actions, are expressed in the rat cerebellum. All these evidences contribute to reinforce the role of diadenosine polyphosphates as signaling molecules in the central nervous system. Finally, we have analyzed the possible differences in the concentration of diadenosine polyphosphates in the cerebellar extracellular medium and changes in the expression levels of their receptors and hydrolyzing enzymes in an animal model of moderate hyperammonemia.

Fig. 1

Detection of Ap_nAs in the cerebellar microdialysis samples. One hundred microliters of cerebellar dialysates were injected in a HPLC system in order to detect and quantify the diadenosine polyphosphates, as described in the “Materials and methods” section. Peaks corresponding to the Ap_nAs were identified by their retention times. Representative chromatograms for the cerebellar microdialysis samples showing the presence of Ap_nAs peaks are shown in a, b, and c (lower traces). The identification of the Ap_nAs was confirmed by injecting the same volume of the samples (100 μL) enriched with 10 pmol of exogenously added Ap₃A (a, upper trace), Ap₄A (b, upper trace), or Ap₅A (c, upper trace) and verifying that the presumptive Ap_nAs peaks coeluted with the corresponding standard

Fig. 2

Concentration of the diadenosine polyphosphates in cerebellar dialysates of hyperammonemic vs control rats. The amounts of Ap₃A (a), Ap₄A (b), and Ap₅A (c) were quantified in microdialysis samples from the cerebellum of rats made hyperammonemic by feeding them with an ammonium supplemented diet (HA) or control animals (Ctrl). Bars represent the mean ± SEM of samples obtained from the cerebellum of 12 (a, b) or 8 (c) different animals

Fig. 3

Rat cerebellum expresses ecto-nucleotide pyrophosphatases/phosphodiesterases and several purinergic receptors susceptible to be activated by diadenosine polyphosphates. a Quantitative real-time PCR for ionotropic P2X1, P2X2, P2X3, and P2X4 receptors, and metabotropic P2Y₁, P2Y₂, P2Y₄, and P2Y₁₂ receptors in rat cerebella. Values were normalized by the content of GAPDH transcript. The results are the mean ± SEM of six animals. b Quantitative real-time PCR for NPP1 and NPP3 ecto-enzymes in rat cerebella. Values were normalized by the content of GAPDH transcript. The results are the mean ± SEM of six animals

Fig. 4

Changes in purinergic receptors expression in the cerebella of hyperammonemic rats. Quantitative real-time PCR for P2X2 and P2Y₄ receptors in control (Ctrl) and hyperammonemic (HA) rat cerebella. Values were normalized by the content of GAPDH transcript. The results are the mean ± SEM of six to seven animals. Statistical significance versus corresponding control, *p < 0.05; **p < 0.01 (Student’s t test)