[ParC-10 cells for modelling parotid gland tissue reorganization]. / Par-C10 sejtek a parotis szöveti szervezodésének modellezésére.

Salivary gland hypofunction, which may occur in head and neck cancers following therapeutic irradiation or in Sjogren's syndrome, drastically impair the patient's quality of life. Conventional treatments do not provide a satisfactory solution to the problem, therefore it is becoming increasingly urgent to develop completely new management approaches in particular, the challenge of restoring the function of acini. Many biologically based interventions studied, thus "reprogramming" with gene therapy of survivor ducts or regeneration potential of progenitor cells in the salivary gland. Our research group has been working on several models, which have shown that by using appropriate media containing extracellular proteins (e.g. BME, basal membrane extract) can be achieved acinar differentiation. A significant proportion of in vitro models of salivary gland are submandibular of origin, which however is different from the development and function of parotid. Our research group aimed to model the potential treatment options for salivary gland hypofunction, the carrier or bioactive molecules directed differentiation, as well as the potential of gene therapy on rat parotid-derived cell line (Par-C10). In our experiments, we have studied the morphological changes of Par-C10 cells cultured on permeable polyester membrane, or in three-dimensional cultures, using varying concentrations of BME. In addition, we have tested the use of recombinant adenovirus vectors that could modify Par-C10 cells and make them useful in gene therapy models. Our data suggest that Par-C10 cell line is suitable for modelling parotid gland tissue organization and may also serve as a useful gene therapy model system.

Bicarbonate transport by the human pancreatic ductal cell line HPAF.

OBJECTIVES: The human pancreatic duct cell line, HPAF, has been shown previously to secrete Cl(-) in response to Ca(2+)-mobilizing stimuli. Our aim was to assess the capacity of HPAF cells to transport and secrete HCO3(-). METHODS: HPAF cells were grown as confluent monolayers on permeable supports. Short-circuit current was measured by voltage clamp. Intracellular pH (pHi) was measured by microfluorometry in cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). RESULTS: In HCO3(-)-free solutions, ATP-evoked changes in short-circuit current were inhibited by bumetanide, and the recovery of pHi from acid loading was abolished by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). In the presence of HCO3(-), ATP-evoked secretion was no longer inhibited by bumetanide, and there was a strong EIPA-insensitive recovery from acid loading, which was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS). ATP, but not forskolin, stimulated HCO3(-) efflux from the cells. CONCLUSIONS: In the absence of HCO3(-), ATP-evoked Cl(-) secretion is driven by a basolateral Na(+)-K(+)-2Cl(-) cotransporter, and pH(i) is regulated by apical and basolateral Na(+)/H(+) exchangers. In the presence of HCO3(-), ATP-evoked secretion is sustained in the absence of Na(+)-K(+)-2Cl(-) cotransporter activity and is probably driven by basolateral Na(+)-HCO3(-) cotransport.

Differential regulation of the apical plasma membrane Ca(2+) -ATPase by protein kinase A in parotid acinar cells.

Cross-talk between intracellular calcium ([Ca(2+)](i)) signaling and cAMP defines the specificity of stimulus-response coupling in a variety of cells. Previous studies showed that protein kinase A (PKA) potentiates and phosphorylates the plasma membrane Ca(2+)-ATPase (PMCA) in a Ca(2+)-dependent manner in parotid acinar cells (Bruce, J. I. E., Yule, D. I., and Shuttleworth, T. J. (2002) J. Biol. Chem. 277, 48172-48181). The aim of this study was to further investigate the spatial regulation of [Ca(2+)](i) clearance in parotid acinar cells. Par-C10 cells were used to functionally isolate the apical and basolateral PMCA activity by applying La(3+) to the opposite side to inhibit the PMCA. Activation of PKA (using forskolin) differentially potentiated apical [Ca(2+)](i) clearance in mouse parotid acinar cells and apical PMCA activity in Par-C10 cells. Immunofluorescence of parotid tissue slices revealed that PMCA1 was distributed throughout the plasma membrane, PMCA2 was localized to the basolateral membrane, and PMCA4 was localized to the apical membrane of parotid acinar cells. However, in situ phosphorylation assays demonstrated that PMCA1 was the only isoform phosphorylated by PKA following stimulation. Similarly, immunofluorescence of acutely isolated parotid acinar cells showed that the regulatory subunit of PKA (RIIbeta) translocated to the apical region following stimulation. These data suggest that PKA-mediated phosphorylation of PMCA1 differentially regulates [Ca(2+)](i) clearance in the apical region of parotid acinar cells because of a dynamic translocation of PKA. Such tight spatial regulation of Ca(2+) efflux is likely important for the fine-tuning of Ca(2+)-dependent effectors close to the apical membrane important for the regulation of fluid secretion and exocytosis.

Vectorial bicarbonate transport by Capan-1 cells: a model for human pancreatic ductal secretion.

Human pancreatic ducts secrete a bicarbonate-rich fluid but our knowledge of the secretory process is based mainly on studies of animal models. Our aim was to determine whether the HCO(3)(-) transport mechanisms in a human ductal cell line are similar to those previously identified in guinea-pig pancreatic ducts. Intracellular pH was measured by microfluorometry in Capan-1 cell monolayers grown on permeable filters and loaded with BCECF. Epithelial polarization was assessed by immunolocalization of occludin. Expression of mRNA for key electrolyte transporters and receptors was evaluated by RT-PCR. Capan-1 cells grown on permeable supports formed confluent, polarized monolayers with well developed tight junctions. The recovery of pH(i) from an acid load, induced by a short NH(4)(+) pulse, was mediated by Na(+)-dependent transporters located exclusively at the basolateral membrane. One was independent of HCO(3)(-) and blocked by EIPA (probably NHE1) while the other was HCO(3)(-)-dependent and blocked by H(2)DIDS (probably pNBC1). Changes in pH(i) following blockade of basolateral HCO(3)(-) accumulation confirmed that the cells achieve vectorial HCO(3)(-) secretion. Dose-dependent increases in HCO(3)(-) secretion were observed in response to stimulation of both secretin and VPAC receptors. ATP and UTP applied to the apical membrane stimulated HCO(3)(-) secretion but were inhibitory when applied to the basolateral membrane. HCO(3)(-) secretion in guinea-pig ducts and Capan-1 cell monolayers share many common features, suggesting that the latter is an excellent model for studies of human pancreatic HCO(3)(-) secretion.