Journal article
Development of a polarized pancreatic ductular cell epithelium for physiological studies
Journal of applied physiology (1985), Vol.125(1), pp.97-106
07/01/2018
DOI: 10.1152/japplphysiol.00043.2018
PMCID: PMC6086968
PMID: 29517421
Abstract
Pancreatic ductular epithelial cells comprise the majority of duct cells in pancreas, control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate (
H
C
O
3
−
) secretion, but are difficult to grow as a polarized monolayer. Using NIH-3T3-J2 fibroblast feeder cells and a Rho-associated kinase inhibitor, we produced well-differentiated and polarized porcine pancreatic ductular epithelial cells. Cells grown on semipermeable filters at the air-liquid interface developed typical epithelial cell morphology and stable transepithelial resistance and expressed epithelial cell markers (zona occludens-1 and β-catenin), duct cell markers (SOX-9 and CFTR), but no acinar (amylase) or islet cell (chromogranin) markers. Polarized cells were studied in Ussing chambers bathed in Krebs-Ringer
H
C
O
3
−
solution at 37°C gassed with 5% CO
2
to measure short-circuit currents (
I
sc
). Ratiometric measurement of extracellular pH was performed with fluorescent SNARF-conjugated dextran at 5% CO
2
. Cells demonstrated a baseline
I
sc
(12.2 ± 3.2 μA/cm
2
) that increased significantly in response to apical forskolin-IBMX (∆
I
sc
: 35.4 ± 3.8 μA/cm
2
,
P
< 0.001) or basolateral secretin (∆
I
sc
: 31.4 ± 2.5 μA/cm
2
,
P
< 0.001), both of which increase cellular levels of cAMP. Subsequent addition of apical GlyH-101, a CFTR inhibitor, decreased the current (∆
I
sc
: 20.4 ± 3.8 μA/cm
2
,
P
< 0.01). Extracellular pH and
H
C
O
3
−
concentration increased significantly after forskolin-IBMX (pH: 7.18 ± 0.23 vs. 7.53 ± 0.19;
H
C
O
3
−
concentration, 14.5 ± 5.9 vs. 31.8 ± 13.4 mM;
P
< 0.05 for both). We demonstrate the development of a polarized pancreatic ductular epithelial cell epithelium with CFTR-dependent
H
C
O
3
−
secretion in response to secretin and cAMP. This model is highly relevant, as porcine pancreas physiology is very similar to humans and pancreatic damage in the cystic fibrosis pig model recapitulates that of humans.
NEW & NOTEWORTHY
Pancreas ductular epithelial cells control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion. Their function is critical because when CFTR is deficient in cystic fibrosis bicarbonate secretion is lost and the pancreas is damaged. Mechanisms that control pancreatic bicarbonate secretion are incompletely understood. We generated well-differentiated and polarized porcine pancreatic ductular epithelial cells and demonstrated feasibility of bicarbonate secretion. This novel method will advance our understanding of pancreas physiology and mechanisms of bicarbonate secretion.
Details
- Title: Subtitle
- Development of a polarized pancreatic ductular cell epithelium for physiological studies
- Creators
- Yunxia O’Malley - Stead Family Department of PediatricsPavana G Rotti - Department of Anatomy and Cell BiologyIan M Thornell - Department of Internal MedicineOriana G Vanegas Calderón - Stead Family Department of PediatricsChristopher Febres-Aldana - The Arkadi M. Rywlin M.D. Department of Pathology and Laboratory MedicineKatelin Durham - Stead Family Department of PediatricsJianrong Yao - Stead Family Department of PediatricsXiaopeng Li - Department of Internal MedicineZheng Zhu - Stead Family Department of PediatricsAndrew W Norris - Stead Family Department of PediatricsJoseph Zabner - Department of Internal MedicineJohn F Engelhardt - Department of Anatomy and Cell BiologyAliye Uc - Stead Family Department of Pediatrics
- Resource Type
- Journal article
- Publication Details
- Journal of applied physiology (1985), Vol.125(1), pp.97-106
- Publisher
- American Physiological Society; Bethesda, MD
- DOI
- 10.1152/japplphysiol.00043.2018
- PMID
- 29517421
- PMCID
- PMC6086968
- ISSN
- 8750-7587
- eISSN
- 1522-1601
- Grant note
- ; ; HL-091842; HL-051670; CA086862 / ; ; DK-084049; DK-097820; DK-096518 / ; ;
- Alternative title
- PANCREATIC DUCTULAR CELL CULTURE
- Language
- English
- Date published
- 07/01/2018
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Pulmonary, Critical Care, and Occupational Medicine; Endocrinology and Diabetes; Anatomy and Cell Biology; Stead Family Department of Pediatrics; Radiation Oncology; Gastroenterology, Hepatology, Pancreatology, and Nutrition; Biochemistry and Molecular Biology; Internal Medicine
- Record Identifier
- 9984024546602771
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