Distribution of P2Y and P2X purinergic receptor expression within the intestine

Abstract

Nucleotides are potent extracellular signaling molecules during homeostasis, infection, and injury due to their ability to activate purinergic receptors. The nucleotide ATP activates P2X receptors (P2RXs), whereas the nucleotides ADP, ATP, UTP, and UDP-glucose selectively activate different P2Y receptors (P2RYs). Several studies have established crucial roles for P2 receptors during intestinal inflammatory and infectious diseases, yet the most extensive characterization of purinergic signaling has focused on immune cells and the central and enteric nervous systems. As epithelial cells serve as the first barrier against irritants and infection, we hypothesized that the gut epithelium may express multiple purinergic receptors that respond to extracellular nucleotide signals. Using the Human Protein Atlas and Gut Cell Survey, we queried single-cell RNA sequencing (RNAseq) data for the P2 purinergic receptors in the small and large intestines. In silico analysis reveals robust mRNA expression of P2RY1, P2RY2, P2RY11, and P2RX4 throughout the gastrointestinal tract. Human intestinal organoids exhibited a similar expression pattern with a prominent expression of P2RY1, P2RY2, and P2RX4, but this purinergic receptor repertoire was not conserved in T84, Caco2, and HT29 intestinal epithelial cell lines. Finally, P2YR1 and P2YR2 agonists elicited robust calcium responses in human intestinal organoids, but calcium responses were weaker or absent in the cell lines. These findings suggest that the gastrointestinal epithelia respond to extracellular purinergic signaling via P2RY1, P2RY2, P2RY11, and P2RX4 receptors and highlight the benefit of using intestinal organoids as a model of intestinal purinergic signaling.NEW & NOTEWORTHY Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, however, these have largely been demonstrated in immune cells and the enteric nervous system. Although epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. This work expands our knowledge of purinergic receptor distribution and relative expression along the intestine.

Keywords: P2X; P2Y; gastrointestinal tract; purinergic receptors.

Graphical abstract

Figure 1.

Human intestinal epithelial cells highly express P2RY1, P2RY2, P2RY11, and P2X4. Single-cell RNAseq data were examined in the human protein atlas for P2Y (A–C) and P2X (D–F) receptors in the epithelium of the small intestine (A and D), colon (B and E), and rectum (C and F). The annotation after each cell type (c-) corresponds to the assigned clustering from the database (ie c-0 is cluster 0).

Figure 2.

Human gastrointestinal epithelial cell expression of P2RY1, P2RY2, and P2RX4 can be independently confirmed in another database. Single-cell RNAseq data were examined in the Gut Cell Survey database for P2Y and P2X receptors in small intestine (A) and large intestine (B).

Figure 3.

Human intestinal and colonic organoids and traditional cell lines have distinct P2 receptor mRNA expression and distribution. A: RNAseq data of human duodenum, jejunum, ileum, and colon organoids. qPCR analysis of monolayers of human jejunal organoids (B), human colonic organoids (C), human T84 cells (D), human HT29 cells (E), and human Caco2 cells (F). Data represented as ΔΔCT normalized to 18S. n = 3–6 biological replicates. Scale bar represents 50 µm.

Figure 4.

Human intestinal organoids respond to P2Y and P2X agonist treatment. A: representative images of agonist treatment in human intestinal organoid monolayers expressing GCaMP6s. B: normalized calcium fluorescence (F/F₀) overtime in vehicle control (black)-, 10 µM ATPγS (purple)-, 10 µM MRS2365 (magenta)-, 10 µM PSB 1114 (teal)-, 100 nM MRS4062 (green)-, or 10 µM MRS2693 (blue)-treated monolayers. C: maximum change in GCaMP6s fluorescence in response to vehicle control (black), 10 µM ATPγS (purple), 10 µM MRS2365 (magenta), 10 µM PSB 1114 (teal), 100 nM MRS4062 (green), or 10 µM MRS2693 (blue) in monolayers. One-way ANOVA. *P < 0.05, ****P < 0.0001. n = 3–5 experiments. Scale bar represents 100 µm.

Figure 5.

Human T84 cells do not respond to P2Y-specific agonists. A: representative images of agonist treatment in human T84 monolayers expressing GCaMP6s. B: normalized GCaMP6s fluorescence (F/F₀) overtime in vehicle control (black)-, 10 µM ATPγS (purple)-, 10 µM MRS2365 (magenta)-, 10 µM PSB 1114 (teal)-, 100 nM MRS4062 (green)-, or 10 µM MRS2693 (blue)-treated monolayers. C: maximum changes in GCaMP6s fluorescence in response to vehicle control (black), 10 µM ATPγS (purple), 10 µM MRS2365 (magenta), 10 µM PSB 1114 (teal), 100 nM MRS4062 (green), or 10 µM MRS2693 (blue) in monolayers. One-way ANOVA. ****P < 0.0001. n = 3–5 experiments. Scale bar represents 100 µm.

Figure 6.

Human HT29 cells do not respond to P2Y-specific agonists. A: representative images of agonist treatment in human HT29 monolayers expressing GCaMP6s. B: normalized GCaMP6s fluorescence (F/F₀) overtime in vehicle control (black)-, 10 µM ATPγS (purple)-, 10 µM MRS2365 (magenta)-, 10 µM PSB 1114 (teal)-, 100 nM MRS4062 (green)-, or 10 µM MRS2693 (blue)-treated monolayers. C: maximum changes in GCaMP6s fluorescence in response to vehicle control (black), 10 µM ATPγS (purple), 10 µM MRS2365 (magenta), 10 µM PSB 1114 (teal), 100 nM MRS4062 (green), or 10 µM MRS2693 (blue) in monolayers. One-way ANOVA. ****P < 0.0001. n = 3–5 experiments. Scale bar represents 100 µm.

Figure 7.

Human Caco2 cells do not respond to P2Y and P2X agonists. A: representative images of agonist treatment in human Caco2 monolayers expressing GCaMP8s. B: normalized GCaMP8s fluorescence (F/F₀) in response to vehicle control (black), 10 µM ATPγS (purple), 10 µM MRS2365 (magenta), 10 µM PSB 1114 (teal), 100 nM MRS4062 (green), or 10 µM MRS2693 (blue) in monolayers. C: maximum changes in GCaMP8s fluorescence in response to vehicle control (black), 10 µM ATPγS (purple), 10 µM MRS2365 (magenta), 10 µM PSB 1114 (teal), 100 nM MRS4062 (green), or 10 µM MRS2693 (blue) in monolayers. One-way ANOVA. n = 3–5 experiments. Scale bar represents 100 µm.