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Am J Physiol Renal Physiol 281: F557-F570, 2001;
0363-6127/01 $5.00
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Vol. 281, Issue 3, F557-F570, September 2001

SPECIAL COMMUNICATION
Neurotransmitter-stimulated ion transport by cultured porcine vas deferens epithelium

Roger L. Sedlacek1, Ryan W. Carlin1, Ashvani K. Singh2, and Bruce D. Schultz1

1 Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506; and 2 Aurora Biosciences, San Diego, California 92121

A collagenase-based dissociation technique has been developed to routinely establish monolayer cultures of freshly isolated porcine vas deferens epithelium. Cells isolated from each tissue are transferred to 25-cm2 tissue culture flasks and grown in a standard cell culture medium. Flasks reach confluency in 3-4 days, and cells are subsequently seeded onto permeable supports. Cultured cells display a monolayer cobblestone appearance and are immunoreactive to anti-ZO-1 and anti-cytokeratin antibodies. Electron microscopy is employed to demonstrate the presence of junctional complexes and microvilli. When evaluated in modified Ussing chambers, cultured monolayers exhibit a basal lumen negative potential difference, high electrical resistance (>1,000 Omega  · cm2), and respond to norepinephrine, vasopressin, ATP, adenosine, and histamine, with changes in short-circuit current indicative of anion secretion. Responses are significantly attenuated in Cl-- and/or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. Attempts to further optimize culture conditions have shown that chronic exposure to insulin increases proliferation rates. Thus the culture method described will reliably produce viable neurotransmitter-responsive cell monolayers that will allow for the characterization of vas deferens epithelial function and associated control mechanisms.

model system; epithelia; anion transport; pH regulation; cystic fibrosis; congenital bilateral absence of the vas deferens


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