Neurotransmitter-stimulated ion transport by cultured porcine vas deferens epithelium.
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-cm(2) 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. cm(2)), 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-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.
Sedlacek
R L
RL
Department of Anatomy and Physiology, Kansas State University, 1600 Denison Ave., Manhattan, KS 66506, USA.
Carlin
R W
RW
Singh
A K
AK
Schultz
B D
BD
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000838:Anions / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006632:Histamine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007365:Intercellular Junctions / Q000502:physiology / Q000648:ultrastructure; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008871:Microvilli / Q000502:physiology / Q000648:ultrastructure; D008954:Models, Biological; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D009638:Norepinephrine / Q000494:pharmacology; D013552:Swine; D014649:Vas Deferens / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D014667:Vasopressins / Q000494:pharmacology
D000838:Anions; D001639:Bicarbonates; D002712:Chlorides; D018377:Neurotransmitter Agents; D014667:Vasopressins; D006632:Histamine; D000255:Adenosine Triphosphate; D000241:Adenosine; D009638:Norepinephrine