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. 2022 Oct 15;11(10):1512.
doi: 10.3390/biology11101512.

Messenger RNA Gene Expression Screening of VIP and PACAP Neuropeptides and Their Endogenous Receptors in Ruminants

Emma Hawley  1 , Kafi Mia  2 , Mustapha Yusuf  2 , Kendall C Swanson  2 , Curt Doetkott  3 , Glenn P Dorsam  1
Affiliations

Messenger RNA Gene Expression Screening of VIP and PACAP Neuropeptides and Their Endogenous Receptors in Ruminants

Emma Hawley et al. Biology (Basel). .

Abstract

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate-Cyclase-Activating Peptide (PACAP) are anti-inflammatory neuropeptides that play important roles in human and rodent gut microbiota homeostasis and host immunity. Pharmacologically regulating these neuropeptides is expected to have significant health and feed efficiency benefits for agriculturally relevant animals. However, their expression profile in ruminant tissues is not well characterized. To this end, we screened for VIP and PACAP neuropeptides and their endogenous GPCRs using 15 different tissues from wethers and steers by RT-qPCR. Our results revealed relatively similar expression profiles for both VIP and PACAP neuropeptide ligands in the brain and intestinal tissue of both species. In contrast, the tissue expression profiles for VPAC1, VPAC2, and PAC1 were more widespread and disparate, with VPAC1 being the most diversely expressed receptor with mRNA detection in the brain and throughout the gastrointestinal tract. These data are an important first step to allow for future investigations regarding the VIP and PACAP signaling pathways in livestock ruminant species.

Keywords: PAC1; PACAP; VIP; VPAC1; VPAC2; ruminant; steer; wether.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Primer Efficiency Curves. Curves made using (A). Bos Taurus and (B). Ovis Aries. Efficiencies are between 91–108%.
Figure A2
Figure A2
Reference Gene Stability Assessment Across Steer and Wether Samples. Average of technical replicates and stability score ranking of reference genes GAPDH, PPIA, B2M and SDHA in (A,B). Steers and (C,D). Wethers.
Figure A3
Figure A3
Coefficient of Variation Between Plates on Separate qPCR Runs. Inter-assay plate control trends with an associated coefficient of variation (C.V.). Data points represent the mean of technical replicates for each plate. Plate # refers to the number of independent qPCR plates performed for this analysis.
Figure 1
Figure 1
cDNA pooling strategy used for species-specific tissues prior to qPCR screening. A total of 15 tissues were harvested from 4 steers and 4 wethers. RNA was extracted from all tissues and used to create a cDNA library. There was a total of 4 biological replicates for each tissue in both species and these cDNAs were pooled into a single sample for qPCR screening of VIP, VPAC1, VPAC2, PACAP, PAC1, and reference genes.
Figure 2
Figure 2
Similar relative mRNA profiles for VIP and PACAP ligands. Data is presented in bar graph form with technical duplicate means +/− SD of pooled cDNA (n = 3 − 4) for indicated tissues measured in (A). wethers or (B). steers. N.D. indicates no detection. (C). Venn diagram representing tissue expression for ligands with overlap indicating identical expression profiles between species.
Figure 3
Figure 3
Differential relative mRNA profiles for VIP and PACAP receptors. Data is presented in bar graph form with technical duplicate means +/− SD of pooled cDNA (n = 3 − 4) for indicated tissues measured in (A). wethers or (C). (C,D). Venn diagrams representing tissue expression of VIP/PACAP receptors with overlap indicating identical expression profiles between (B). wethers and (D). steers.
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