Abstract

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Na_v1.9 in mediating murine responses. The application of UTP (P2Y₂ and P2Y₄ agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y₁, P2Y₁₂, and P2Y₁₃ agonist) also increased action potential firing, an effect blocked by the selective P2Y₁ receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y₁ and P2Y₂ transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Na_v1.9 transcripts colocalized in 86% of P2Y₁-positive and 100% of P2Y₂-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na_v1.9^−/− mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.

SIGNIFICANCE STATEMENT Chronic visceral pain is a debilitating symptom of many gastrointestinal disorders. The activation of pain-sensing nerves located in the bowel wall and their sensitization to physiological stimuli, including bowel movements, underpins the development of such pain, and is associated with mediators released during disease. This work addresses the unstudied role of purine and pyrimidine nucleotides in modulating colonic nociceptors via P2Y receptors using a combination of electrophysiological recordings from human ex vivo samples and a detailed functional study in the mouse. This is the first report to identify colonic purinergic signaling as a function of P2Y receptor activation, in addition to established P2X receptor activity, and the results contribute to our understanding of the development of visceral pain during gastrointestinal disease.