Fig 2: Extracellular succinate stimulates SUCNR1-dependent SMG contraction.A) Representative reflected light image of SMGs in newborn pig trachea after cartilage and smooth muscle layers were removed.B) Representative images of SMGs (EPCAM+, red) before and after succinate treatment. Closed lines outline the selected SMG regions for area measurement.C) Summary of SMG area treated with succinate or methyl succinate during live imaging of SMGs. 3 glands each group.D, E) Quantification of succinate-induced SMG contraction. Relative SMG area 3 minutes after a treatment. Panel D, SMGs were treated with 10 mM methyl succinate or succinate. Panel E, Airway surface epithelia were removed. SMGs were pre-incubated with or without SUCNR1 antagonist NF-56-EJ40 (5 µM) and induced with succinate. Data points are the mean of 3 SMGs from each pig; p values are shown in graph; p > 0.05 is considered as not significant (n.s), Student’s t test.F, G) Succinate prevents SMG swelling due to mucus secretion triggered by substance P. Panel F, representative images of SMGs that were pre-incubated with or without NF-56-EJ40 and treated with substance P and/or succinate. Panel G, quantification of SMG swelling response to substance P and/or succinate treatment. P values are shown in graph, n = 5 pigs, paired Student’s t test.H) Quantification of acinar cell shrinkage under succinate and/or substance P stimulation. P = 0.0006, n = 5 pigs, one way ANOVA. See also Figure S5.

Fig 3: ATP-P2Y1R signaling controls myoepithelial cell contraction in SMGs.A) Characterization of cultured primary myoepithelial cells after 5 days of culture. aSMA and KRT5 are markers for myoepithelial cells.B) Representative images of primary myoepithelial cells (KRT5+, red) stained with P2Y1 receptor antibody (gray).C) Representative Fura-2 ratiometric Ca2+ images of primary myoepithelial cells before and after ATP.D) Summary of ratiometric Ca2+ imaging (R/R0, R = 340/380) in primary myoepithelial cells. Cells were pre-incubated with or without P2Y1 receptor antagonist MRS2500 (5 µM). Dashed line indicates the time when ATP (1, 10, 100 µM) was added. n = 3 pigs.E, F) ATP stimulates primary myoepithelial cell contraction (presented as ratio of total area after treatment). Panel E, effects of 5-HT, CGRP, or ATP on primary myoepithelial cell contraction. Panel F, P2Y1 receptor antagonist MRS2500 abolished ATP controlled primary myoepithelial cell contraction. Each data point represents the mean of imaged cells from a pig; p values are shown in graph; n.s, not significant; *, p < 0.05; one sample t test for panel E, paired Student’s t test for panel F.G) WT and CF SMG contraction with ATP (1 mM) or succinate (10 mM) treatment ex vivo. Each data point is the mean of 3 SMGs from an individual pig. P values are shown in graph, paired Student’s t test.H) Model of SMG PNEC sensing succinate to trigger myoepithelial contraction. PNECs located in airway SMGs express SUCNR1, the receptor for succinate. Under hypoxia, infection, and inflammation, succinate is generated by macrophages, other epithelial cells, or microorganisms in airway surface liquid, which diffuses into the SMGs. SUCNR1 on SMG PNEC responds to extracellular succinate by releasing ATP to the basolateral environment. ATP activates P2Y1 receptors on myoepithelial cells to cause Ca2+-dependent myoepithelial contraction. See also Figure S7.

Fig 4: Porcine and human PNECs express SUCNR1.A) G protein-coupled receptors in SMG scRNA-seq data (Yu et al., 2022). Genes are ranked by false discovery rate (FDR) from the lowest to the highest. Ser, serous cell; Myoep, myoepithelial cell; Muc, mucous cell. See also Table S1.B-D) Characterization of SUCNR1 expression using smFISH in porcine PNECs. B is representative smFISH images of SUCNR1, ASCL1, and POU2F3 in SMGs and airway surface epithelia (ASE). C is the percentage of cells that are positive for SUCNR1 for PNECs (224 cells) and tuft cells (88 cells). n = 4 pigs (2WT, 2CF); p < 0.0001, paired Student’s t test. D is normalized SUCNR1 level (smFISH counts) in WT and CF PNECs (224 cells). P value was calculated based on individual SMGs; p > 0.05 is considered as not significant (n.s), unpaired Student’s t test.E) Apical distribution of SUCNR1 in airway surface epithelia. Newborn pig trachea was infected with lentivirus carrying CMV promoter driven Myc-tagged SUCNR1 cDNA. After one week of ex vivo culture, tissues were sectioned and stained with anti-Myc antibody. Dashed line highlights airway surface, arrowheads point to cells with apical Myc tag staining.F) Representative immunofluorescent images of neuron fibers (SYP+, green) and PNECs (KRT20+, gray) in porcine SMGs (EPCAM+, red). Arrowhead highlights the proximity of PNECs and neurons.G) Uniform manifold approximation and projection (UMAP) of pig, human, and mouse airway rare cell types grouped by cell-type (left) and organism (right).H) Expression of PNEC markers (ASCL1, INSM1) in UMAP of pig, human, and mouse airway rare cell types.I) Expression of SUCNR1 in the PNEC cluster of human, pig, and mouse airway scRNA-seq data.J) Percentage of SUCNR1+ cells in PNECs and tuft cells (tuft1 plus tuft2) in human, pig, and mouse airway scRNA-seq datasets. Data points represent biological samples from the original scRNA-seq datasets. P values are shown in graph, unpaired Student’s t test.K) Comparison of PNEC markers between human and pig vs. mouse. Marker genes for both human and pig PNECs are shown. LogFC, fold change at natural log scale. See also Figure S3.