PVD9902, a porcine vas deferens epithelial cell line that exhibits neurotransmitter-stimulated anion secretion and expresses numerous HCO3(-) transporters.
Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ohms x cm2), confluent monolayers that responded to forskolin with an increase in short-circuit current (I(sc); 8 +/- 1 microA/cm2) that required Cl-, HCO3(-), and Na+, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na+-HCO3(-) cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl-/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I(sc). Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I(sc). RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the alpha- and gamma-subunits of the epithelia Na+ channels (alpha- and gamma-ENaC), but not beta-ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.
Carlin
Ryan W
RW
Dept. of Anatomy and Physiology, Kansas State Univ., 1600 Denison Ave., Manhattan, KS 66506, USA.
Sedlacek
Roger L
RL
Quesnell
Rebecca R
RR
Pierucci-Alves
Fernando
F
Grieger
David M
DM
Schultz
Bruce D
BD
D000818:Animals; D000838:Anions / Q000378:metabolism*; D002460:Cell Line / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism*; D007150:Immunohistochemistry; D008297:Male; D018377:Neurotransmitter Agents / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism*; D013552:Swine; D014649:Vas Deferens / Q000166:cytology* / Q000378:metabolism
D000838:Anions; D018377:Neurotransmitter Agents; D027982:Sodium-Bicarbonate Symporters; D005576:Colforsin