Functional reconstruction of lost tissue by regenerative
therapy of salivary glands would be of immense benefit following
radiotherapy or in the treatment of
Sjogren's syndrome. The purpose of this study was to develop primary cultures of human salivary gland cells as potential regenerative resources and to characterize their acinar/ductal phenotype using electrophysiological measurements of ion transport. Human salivary gland cultures were prepared either from adherent submandibular gland cells (huSMG) or from mixed adherent and nonadherent cells (PTHSG) and were cultivated in Hepato-STIM or minimum essential medium (MEM). Expression of key epithelial marker
proteins was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Transepithelial electrical resistance (TER) was monitored following seeding the cells on Transwell membranes. Transepithelial ion transport was estimated by short-circuit current (Isc) measurements in an Ussing chamber. Both huSMG and PTHSG cells showed epithelial characteristics when cultivated in Hepato-STIM, while fibroblast-like elements dominated in MEM. Compared to intact tissue, cultivation of the cells resulted in substantial decreases in AQP5 and NKCC1 expression and moderate increases in
claudin-1 and ENaC expression. Both cultures achieved high TER and transepithelial
electrolyte movement in Hepato-STIM, but not in MEM. The Isc was substantially reduced by basolateral Cl(-) and
bicarbonate withdrawal, indicating the involvement of basolateral-to-apical
anion transport, and by the blockade of apical ENaC by
amiloride, indicating the involvement of apical-to-basolateral Na(+) transport. An almost complete inhibition was observed following simultaneous ENaC block and withdrawal of the two
anions. Isc was enhanced by either apical
adenosine triphosphate (
ATP) or basolateral
carbachol application, but not by
forskolin, confirming the expected role of Ca(2+)-activated regulatory pathways in
electrolyte secretion. Inhibition of basolateral NKCC1 by
bumetanide reduced the response to
ATP, indicating the active involvement of this transporter in Cl(-) secretion. In conclusion, we have demonstrated that both PTHSG and huSMG primary cultures cultivated in Hepato-STIM form two-dimensional monolayers in vitro on permeable supports and achieve active vectorial transepithelial
electrolyte transport. The presence of both basolateral-to-apical
anion fluxes and an apical-to-basolateral Na(+) flux indicates both acinar and ductal characteristics. With further refinement, this model should provide a firm basis for new interventions to correct salivary gland dysfunction.