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

Abstract

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...

Gastrin receptors on isolated canine parietal cells.

“…1 A). Canine fundic mucosal cells were separated by elutriation into nine fractions by using a protocol previously described (22). Stepwise density-gradient separation of enriched parietal and chief cell elutriator functions.…”

“…Two fractions were collected: fraction A banding above the 65% heavy step and fraction B that pelleted through this 65% heavy solution. The parietal cell and chief cell contents were determined counting slides prepared on a cytocentrifuge and stained with periodic acid Schiff (22). and the chief cells enriched to 76 and 77%, respectively.…”

“…In brief, the fundic mucosa from dogs killed with a pentobarbital infusion was bluntly separated from the submucosa and then sequentially incubated in crude collagenase (0.25 mg/ml) and EDTA (1 mM). Cell separation was performed by using a elutriator rotor (Beckman Instruments, Inc., Fullerton, CA) (8,22). This technique separates cells on the basis of varying sedimentation velocity, which is proportional to the square of the cell radius.…”

“…This technique separates cells on the basis of varying sedimentation velocity, which is proportional to the square of the cell radius. Fractions were collected according to the protocol outlined previously (22).…”

Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor alpha and insulinlike growth factor I.

“…Material retained on the second filter was suspended in HBSS containing 0.1 mg/ml dithiothreitol and 0.01 mg/ml DNase. This cell suspension was elutriated primarily to eliminate bacteria (15). Cells (2-3 X 108) were loaded into a model J 5.0 elutriator rotor (Beckman Instruments, Inc., Fullerton, CA) at a flow rate of 87.5 ml/min and an rpm of2,000.…”