Mouse mammary epithelial cells on floating collagen gels: Transepithelial ion transport and effects of prolactin

182

The tracheal mucosa from a 12-year-old girl was digested with collagenase 4 hr after her death from cystic fibrosis. Forty million viable cells were obtained. The cells, plated at 106 per cm2 onto four Nuclepore filters coated with human placental collagen, formed confluent monolayers after 1 day. Their ultrastructure was similar to that of normal human cells. They were studied in conventional Ussing chambers or with intracellular microelectrodes on days 5-7 after plating. The monolayers displayed resistance of 380 ± 50 fkcm2 and short-circuit current (Isc) of 1.8 ± 0.4 ItAAcm 2.This resistance is similar to that obtained for dog or normal human monolayers. The 4c is less than normal human (-3 IAAcm 2) or dog ('10 ,uAAcm-2) cells. The cystic fibrosis cells resembled normal Inonolayers in that serosal ouabain and mucosal amiloride inhibited Is, while mucosal ouabain or serosal amiloride had no effect. They differed from normal human or dog cells in that Ic was not inhibited by bumetanide and the stimulation of Ic by isoproterenol or prostaglandin E2 was greatly reduced or abolished. Addition of isoproterenol depolarized apical membrane potential (ia) and decreased fractional resistance (fR) in normal human and dog but had no effect on ia or fR in cystic fibrosis cells. Reduction of mucosal chloride from 120 to 5 mM by replacement with gluconate increased fR of dog and normal human monolayers and depolarized l,, by 22 (dog) or 30 (human) mV. In cystic fibrosis monolayers, chloride replacement hyperpolarized *la by 2 mV and had little effect on fR. These results suggest that the primary defect in cystic fibrosis is reduced apical membrane chloride conductance.

mentioning

confidence: 99%

Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium.

Widdicombe¹,

Welsh²,

Finkbeiner³

1985

182

“…Because of this complicating factor, we determined the effect of terbutaline, not on the potential difference, but on the apparent short-circuit current, where short-circuit current is given by the ratio of open-circuit potential difference to transepithelial resistance. Terbutaline was found to increase short-circuit current by 28%, from 3. (25,34,35) and LLC-PK1 renal cells (22) but less than those of mammary cells on floating collagen gels (36) and toad bladder cells (37). As expected from studies of epithelial cells in vitro, amiloride was most active when it was added to the apical side and ouabain was most active when it was added to the basal side of the monolayer (38,39).…”

mentioning

confidence: 99%

Transepithelial transport by pulmonary alveolar type II cells in primary culture.

Mason¹,

Williams²,

Widdicombe³

et al. 1982

234

Fluid and electrolyte transport by epithelial cells in vitro can be recognized by the ability of cultured cells to form domes and by the electrical properties of monolayer cultures. Pulmonary alveolar epithelial cells are thought to be partially responsible for fluid movement in the fetal lung, but their role in electrolyte transport in the adult lung is not known. We isolated alveolar type II cells from adult rat lung and maintained them on plastic culture dishes alone, on plastic culture dishes coated with an extracellular matrix, and on collagen-coated Millipore filters. Numerous large domes were formed on culture dishes coated with the extracellular matrix; smaller domes were formed on uncoated plastic culture dishes. Sodium butyrate (3 mM) stimulated dome formation. Transmission electron microscopy showed that the epithelial cells had flattened but still retained lamellar inclusions and that the cells were polarized with microvilli on the apical surface facing the culture medium. The electrical properties of the monolayers maintained on collagen-coated Millipore filters were tested in two laboratories. The transepithelial potential differences were 0.7 ± 0.1 mV (24 filters, seven experiments) and 1.3 ± 0.1 mV (13 filters, two experiments) apical side negative, and the corresponding resistances were 217 + 11 ohm.cm2 and 233 ± 12 ohm.cm2. Terbutaline (10 FM) produced a biphasic response with a transient decrease and then a sustained increase in potential difference. Amiloride (0.1 mM) completely abolished the potential difference when it was added to the apical side but not when it was added to the basal side, whereas 1 mM ouabain inhibited the potential difference more effectively from the basal side. Thus, type II cells form a polarized epithelium in culture, and these cells actively transport electrolytes in vitro.The alveolar space of adult mammalian lungs is lined by only a thin film of fluid. Although the amount offluid is not known precisely, one estimate is that 20 ml of alveolar fluid is distributed over a surface area ofabout 70 m2 (1). When lungs are fixed for electron microscopy by vascular perfusion, the only areas that appear to contain fluid are the corners ofthe alveoli, where the radius ofcurvature is short (2). At the corners, the net force of surface tension, which is a function of the radius ofcurvature and the actual surface tension, is directed to draw fluid into the alveolus (3-7). How this force is counterbalanced is not known. Although most investigators have tended to discount the possibility of active transport across alveolar epithelium (7, 8), recent observations in vivo suggest that there may be active transport of fluid from the airspace into the interstitium (9, 10).Cell culture techniques have been used to study transepithelial fluid movement. Epithelial cells that transport fluid form domes or hemicysts in culture (11,12

“…In previous studies the electrochemical properties of monolayers in culture have been characterized for continuous cell lines derived from dog kidney (3,4), pig (5), and amphibian urinary bladder (6) and for primary cultures from dispersed mouse mammary glands (7). Monolayers have been formed previously from dispersed, unfractionated rabbit gastric mucosal cells (15), although fetal tissue was used to avoid the problems with bacterial contamination encountered when using adult gastric mucosa.…”

Section: Resultsmentioning

confidence: 99%

Electrical effects of histamine on monolayers formed in culture from enriched canine gastric chief cells.

Ayalon¹,

Sanders²,

Thomas³

et al. 1982

To develop techniques for studying transport properties and secretory function of selected cell types in the gastric mucosa, separated fractions of dispersed canine fundic mucosal cells were placed in short-term culture to form epithelial monolayers. Cell fractions enriched in either chief, parietal, or mucous cells were prepared by using counterflow centrifugation and were plated on type I collagen. An epithelial monolayer formed by ==36 hr. Immunofluorescence with an antipepsinogen I antibody revealed pepsinogen-containing granules in >95% of the cells, regardless ofwhether the monolayers were formed from the mucous, chief, or parietal cell-enriched fractions. Upon achieving confluency, chief cell monolayers were mounted in Ussing chambers to study their electrical properties. Under basal conditions, monolayers (n = 6) had a spontaneous potential difference (PD) (±SEM) of 26 ± 4 mV (apical surface negative), a short-circuit current (Isc) (±SEM) of 16 ± 2 ,LA/cm2, and a transepithelial resistance (R) (± SEM) of 1,480 -210 fl-cm2 Histamine increased the short-circuit current, an effect blocked by an H2-receptor antagonist. Seventy percent of the spontaneous PD was amiloride sensitive, suggesting sodium absorption accounted for a major component of the PD. These preparative techniques yield highly enriched chiefcell monolayers, which maintain morphological and functional cellular differentiation for >48 hr in culture, thus allowing study oforiented functions ofa selected cell type. The present studies indicate that an H2 receptor enhances electrogenic ion transport in chief cell monolayers, indicating that histamine can act on fundic mucosal cells other than just parietal cells.The characterization of transport properties of selected cell types in tissues with heterogeneous cell populations such as the gastric mucosa is ofgreat interest to epithelial physiologists. The gastric mucosa has two types of mucous cells-surface mucous cells that line the lumen of the stomach and mucous neck cells that occupy the upper portion of the gastric glands. The gastric glands, found in the fundic region ofthe stomach, are composed primarily of the acid-secreting parietal cells and the chief cells, which secrete the acid protease pepsin. Elucidation of the role ofspecific cell types in the varied functions ofthe gastric mucosa has proven to be a difficult problem. The difficulty has been encountered not only because of the cellular heterogeneity of the mucosa but also because the regulatory pathways that modulate cell function are quite complex, with many ofthe chemical transmitters involved present within the mucosa itself. Thus, the cellular mechanisms regulating ion transport, the secretory function ofparietal, chief, mucous, and endocrine cells, and the mechanisms involved in gastric mucosal defense against ulcer formation have not been clarified in studies with intact mucosa.Tissue dispersion and cell separation techniques (1, 2) circumvent some of the problems that result from the heterogeneity of cell types and th...