A primary colonic crypt model enriched in enteroendocrine cells facilitates a peptidomic survey of regulated hormone secretion.
To enable the first physiologically relevant peptidomic survey of gastrointestinal tissue, we have developed a primary mouse colonic crypt model enriched for enteroendocrine L-cells. The cells in this model were phenotypically profiled using PCR-based techniques and showed peptide hormone and secretory and processing marker expression at mRNA levels that were increased relative to the parent tissue. Co-localization of glucagon-like peptide-1 and peptide YY, a characteristic feature of L-cells, was demonstrated by double label immunocytochemistry. The L-cells displayed regulated hormone secretion in response to physiological and pharmacological stimuli as measured by immunoassay. Using a high resolution mass spectrometry-based platform, more than 50 endogenous peptides (<16 kDa), including all known major hormones, were identified a priori. The influence of culture conditions on peptide relative abundance and post-translational modification was characterized. The relative abundance of secreted peptides in the presence/absence of the stimulant forskolin was measured by label-free quantification. All peptides exhibiting a statistically significant increase in relative concentration in the culture media were derived from prohormones, consistent with a cAMP-coupled response. The only peptides that exhibited a statistically significant decrease in secretion on forskolin stimulation were derived from annexin A1 and calcyclin. Biophysical interactions between annexin A1 and calcyclin have been reported very recently and may have functional consequences. This work represents the first step in characterizing physiologically relevant peptidomic secretion of gastrointestinally derived primary cells and will aid in elucidating new endocrine function.
Nikoulina
Svetlana E
SE
Amylin Pharmaceuticals, Inc., San Diego, California 92121, USA.
Andon
Nancy L
NL
McCowen
Kevin M
KM
Hendricks
Michelle D
MD
Lowe
Carolyn
C
Taylor
Steven W
SW
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D003106:Colon / Q000166:cytology* / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D019858:Enteroendocrine Cells / Q000166:cytology* / Q000378:metabolism; D005768:Gastrointestinal Hormones / Q000737:chemistry / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology* / Q000378:metabolism; D055442:Metabolome; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013048:Specimen Handling / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003470:Culture Media; D005768:Gastrointestinal Hormones; D010455:Peptides