Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut

Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD⁺ and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca²⁺-activated small-conductance K⁺ (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1^−/− mice. Up4A induced P2Y1R–SK-channel–mediated hyperpolarization in isolated PDGFRα⁺ cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca²⁺ transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD⁺, or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.Uridine adenosine tetraphosphate (Up4A) is, to the authors’ knowledge, the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. Up4A is a recently-identified, nonpeptide, endothelium-derived vasoconstrictor (1, 2). Up4A is likely associated with blood pressure regulation, because its levels in plasma are elevated in hypertensive subjects (3) and it causes vasoconstriction in deoxycorticosterone acetate-salt hypertensive rats (4) and type 2 diabetic rats (5). Up4A also contracts rat and human airways (6) and rat gastric smooth muscles (7). Pharmacological studies suggest that Up4A causes vasoconstriction via activation of P2X1, P2Y2, and P2Y4 receptors (1) and endothelium-dependent vasodilatation via activation of P2Y1 and P2Y2 receptors (8). In porcine coronary artery Up4A causes vasodilatation via adenosine (P1) receptors (9). Furthermore, Up4A causes vascular smooth muscle cell proliferation and migration (10), stimulates monocyte and lymphocyte oxidative burst activities (11), is a potent proinflammatory agent in the vascular wall (12), and may contribute to the proinflammatory status in patients with chronic kidney disease (11). Plasma of healthy human subjects contains ∼50 nmol/L Up4A, which is sufficient to elicit vascular effects (1). The role of Up4A in the gastrointestinal (GI) tract is unknown.Enteric neural regulation of GI motility includes motor neurotransmission mediated by inhibitory neurons releasing purines that act via P2Y1 receptors (P2Y1Rs) (13–17) and apamin-sensitive small-conductance Ca²⁺-activated K⁺ (SK) channels (13, 14, 18, 19). ATP (20), NAD⁺ (14, 15, 21), and adenosine 5′-diphosphate ribose (ADPR) (17) activate P2Y1R and SK channels and might be inhibitory neurotransmitters in the colon (22). Because Up4A appears to stimulate P2Y1Rs in endothelium, and P2Y1Rs are important for purinergic signaling in the colon, we investigated whether Up4A is released in colonic muscle, whether Up4A affects membrane potentials and contractions of colonic muscles, whether Up4A is an agonist for P2Y1R, whether cells expressing PDGF receptor α (PDGFRα) are targets of Up4A, and how Up4A is metabolized in colons of humans and mice.