Elsevier

Neuropharmacology

Volume 91, April 2015, Pages 77-86
Neuropharmacology

Evidence for the existence of pyrimidinergic transmission in rat brain

https://doi.org/10.1016/j.neuropharm.2014.12.019Get rights and content

Highlights

  • The uridine nucleotides UTP and UDP are detected in brain ECF of rats under basal conditions.

  • ECF concentrations of UTP and UDP can be enhanced by exogenous uridine administration.

  • Neuronal activity enhances while blockade of depolarization reduces these concentrations.

  • Enhanced extracellular UTP is associated with increased cholinergic neurotransmission.

  • P2Y2 receptors mediate this effect.

Abstract

The uridine nucleotides uridine-5′-triphosphate (UTP) and uridine-5′-diphosphate (UDP) have previously been identified in media from cultured cells. However, no study to date has demonstrated their presence in brain extracellular fluid (ECF) obtained in vivo. Using a novel method, we now show that UTP and UDP, as well as uridine, are detectable in dialysates of striatal ECF obtained from freely-moving rats. Intraperitoneal (i.p.) administration of uridine or exposure of striatum to depolarizing concentrations of potassium chloride increases extracellular uridine, UTP and UDP, while tetrodotoxin (TTX) decreases their ECF levels. Uridine administration also enhances cholinergic neurotransmission which is accompanied by enhanced brain levels of diacylglycerol (DAG) and inositol trisphosphate (IP3) and blocked by suramin, but not by PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) or MRS2578 suggesting a possible mediation of P2Y2 receptors activated by UTP. These observations suggest that uridine, UTP and UDP may function as pyrimidinergic neurotransmitters, and that enhancement of such neurotransmission underlies pharmacologic effects of exogenous uridine on the brain.

Introduction

Uridine, the principal circulating pyrimidine nucleoside in humans (Wurtman et al., 2000) and its nucleotide products uridine-5′-diphosphate (UDP), uridine-5′-triphosphate (UTP), UDP-glucose, UDP-galactose (Lecca and Ceruti, 2008) and cytidine-5′-triphosphate (CTP) (Cansev et al., 2005) affect numerous physiological functions, including the syntheses of RNA (Lecca and Ceruti, 2008) and membrane phosphatides (Ulus et al., 2006, Wurtman et al., 2006, Cansev and Wurtman, 2007, Sakamoto et al., 2007), and the differentiation of neuron-related cells (e.g. neurite outgrowth) (Pooler et al., 2005). Uridine's effects on phosphatide synthesis are mediated by CTP, an intermediate in the Kennedy Cycle (Kennedy and Weiss, 1956), while its stimulation of neuronal differentiation (Pooler et al., 2005) involves the activation by UTP of brain P2Y2 receptors (Lustig et al., 1993, Pooler et al., 2005).

The uridine nucleotides UTP and UDP are ligands to several P2Y receptors (Abbracchio et al., 2006). UTP activates P2Y2 (Lustig et al., 1993) and P2Y4 (Communi et al., 1995) receptors while UDP activates P2Y6 (Communi et al., 1996) and P2Y14 receptors (Carter et al., 2009). Activation of these cell surface receptors in the brain requires that UTP and UDP are released from cells into the extracellular fluid (ECF) (Cansev, 2007, Lecca and Ceruti, 2008). Therefore, intensive work has been done to identify uridine nucleotides in brain ECF. Although previous in vitro studies reported the detection of UTP in the culture medias of a variety of cells in low nanomolar ranges (1 ± 10 nM in 0.5 ml medium bathing 2.5 cm2 dish) (Lazarowski et al., 1997, Lazarowski and Harden, 1999), to date, only uridine (Dobolyi et al., 1998), but not UTP or UDP, has been identified in brain ECF in vivo.

Therefore, the aim of the present study was to detect and quantify uridine and its nucleotides UTP and UDP in brain ECF by in vivo microdialysis. We initially aimed to solve the possible problem of rapid hydrolysis of extracellular nucleotides by membrane-bound ecto-nucleotidase enzymes (Zimmermann, 1996) which might have been the reason for prior failure to detect uridine nucleotides in brain ECF in vivo. We have thus developed a method – based on blocking the ecto-nucleotidase enzymes that would destroy the UTP and UDP in ECF dialysates – for quantifying these nucleotides in brain ECF.

Using that method, we confirmed their presence in rat brain ECF in vivo and observed increases in their levels following intraperitoneal (i.p.) administration of uridine in doses known to raise (Cansev et al., 2005) plasma and brain uridine levels and brain levels of UTP and UDP. We further observed that uridine, UTP and UDP are released from excitable cells in experimental designs including potassium chloride-induced neuronal depolarization and tetrodotoxin (TTX) blockade of action potentials. The enhanced release of these pyrimidines subsequently enhanced cholinergic neurotransmission, manifested by increased acetylcholine release; this effect was blocked by suramin, a non-selective antagonist of P2Y2 and P2Y6 receptors, but not by PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid), a non-selective antagonist of P2Y4 and P2Y6 receptors or MRS2578, a selective antagonist of P2Y6 receptors.

To the best of our knowledge, our data provide the first comprehensive evidence for a pyrimidinergic neurotransmission, the enhancement of which enhances cholinergic neurotransmission via, probably, P2Y2 receptors.

Section snippets

Animals

Adult male Sprague–Dawley rats (300–350 g; Experimental Animals Breeding and Research Center, Uludag University Medical School, Bursa, Turkey) were housed in groups of four in a temperature controlled room with free access to standard rat chow and water under a 12 h light/dark cycle. The experimental protocol was approved by the Animal Care and Use Committee of Uludag University, Bursa, Turkey (Approval ID: 2008-12/7), and all experiments conformed to the National Institutes of Health Guide for

Effects of exogenous uridine on serum and brain uridine, and brain UTP and UDP levels

In initial experiments, the ability of various i.p. doses of uridine to raise serum uridine concentrations in rats was investigated. Mean basal serum uridine concentrations analyzed at zero time points were 2.4 ± 0.3 μM. These concentrations were increased significantly 1 h after i.p. injection of 1 or 0.5 mmol/kg uridine, to 36.5 ± 0.9 μM (15.2 fold) or 19.7 ± 1.7 μM (8.2 fold) respectively (p < 0.001 for each), but not after i.p. injection of 0.1 mmol/kg uridine (Fig. 1A). These increases

Discussion

These data provide the first evidence that the uridine nucleotides UTP and UDP exist in brain ECF under baseline conditions and that their concentrations can be enhanced by administering uridine systemically. Stimulation of excitable brain cells by potassium chloride enhances the release of uridine, UTP and UDP, while on the other hand, blocking neuronal depolarization by TTX reduces their extracellular concentrations. Hence, the three pyrimidines are secreted constitutively and, to a greater

Conclusion

In conclusion, our data show, for the first time, that pyrimidinergic neurotransmission occurs in brain, mediated by the basal and depolarization-induced release of uridine, UTP and UDP into the extracellular space, and by probable subsequent activation of P2Y2 receptors by UTP to mediate enhanced cholinergic neurotransmission. These data open new avenues for pyrimidine research and may contribute to our understanding of pathogenic mechanisms underlying Alzheimer's disease.

Acknowledgments

The present study was funded in part by The Scientific and Technological Research Council of Turkey (Grant# 108S354) and the Turkish Academy of Sciences (GEBIP Scholarship, 2009). We thank Ant Teknik Company (Istanbul, Turkey) for performing LC-MS/MS analyses and Prof. Levent R. Buyukuysal for his kind gift of TTX.

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