Neuropeptide S receptor induces neuropeptide expression and associates with intermediate phenotypes of functional gastrointestinal disorders

Abstract

Background & aims: NPSR1, the receptor for neuropeptide S (NPS), is expressed by gastrointestinal (GI) enteroendocrine cells, and is involved in inflammation, anxiety, and nociception. NPSR1 polymorphisms are associated with asthma and inflammatory bowel disease. We aimed to determine whether NPS induces expression of GI neuropeptides; and to associate NPSR1 single nucleotide polymorphisms (SNPs) with symptom phenotype and GI functions in health and functional GI disorders (FGID).

Methods: The effect of NPS on messenger RNA expression of neuropeptides was assessed using real-time polymerase chain reaction in NPSR1-tranfected HEK293 cells. Seventeen NPSR1 SNPs were successfully genotyped in 699 subjects from a regional cohort of 466 FGID patients and 233 healthy controls. Associations were sought using gender-adjusted regression analysis and false discovery rate correction.

Results: NPS-NPSR1 signaling induced increased expression of cholecystokinin, vasoactive intestinal peptide, peptide YY, and somatostatin. There were no significant associations with phenotypes of FGID symptoms. There were several NPSR1 SNPs associated with individual motor or sensory functions; the associations of SNPs rs2609234, rs6972158, and rs1379928 with colonic transit rate remained significant after false discovery rate correction. The rs1379928 polymorphism was also associated with pain, gas, and urgency sensory ratings at 36 mm Hg distention, the level prespecified for formal testing. Associations with rectal sensory ratings were not significant after false discovery rate correction.

Conclusions: Expression of several neuropeptides is induced upon NPS-NPSR1 signaling; NPSR1 variants are associated with colonic transit in FGID. The role of the NPS system in FGID deserves further study.

Figure 1. NPSR1 is expressed in intestinal enteroendocrine cells

Immunohistochemistry stainings performed with an affinity purified NPSR1-specific antibody (HPA007489) on human normal intestinal tissue, reported in the Human Protein Atlas portal (http://www.proteinatlas.org). Full size images and additional information are publicly available at the website, hosted by the Swedish Human Proteome Resource (HPR) program.

Figure 2. Effect of NPS on neuropeptides expression

HEK293 cells were transfected in duplicate experiments with NPSR1 cDNA or empty vector (ctrl) and stimulated with 1 μM NPS for six hours. mRNA levels for each gene were measured by quantitative Real-Time PCR and are expressed as relative fold induction (+SD), compared to control cells. CCK = cholecystokinin; CALCA = calcitonin-related polypeptide alpha; GHRL = ghrelin; MLN = motilin; PYY = peptide YY; PPY = pancreatic polypeptide; SST = somatostatin; VIP = vasoactive intestinal peptide. nd = not detected.

Figure 3. LD map and characteristics of the studied NPSR1 SNPs

Left: LD and haplotype block structure (LD structure) obtained from Haploview 4 analysis of genotyping data. The numbers in each box correspond to LD coefficient D’ between respective SNPs (only values for LD <100% are reported). Center: SNPs (marker) are listed with alleles at each locus (SNP - minor allele in lower case) and minor allele frequency in controls (MAF ctrl). Right: position of each SNP within then NPSR1 coding region (gene region), and corresponding effect on mRNA (mRNA).