S. Ito

Requirements for Restitution of the Surface Epithelium of Frog Stomach After Mucosal Injury

In frog fundic mucosae mounted in Ussing chambers, exposure to luminal 1 M NaCl for 10 min caused a sharp immediate decrease in potential difference, resistance, short circuit current, and acid secretion, but within 4-6 h these readings had returned toward control values. After initial severe destruction of surface epithelial cells, gradual morphologic restitution occurred within 4-6 h. A Ca2+-free nutrient solution and 4 mM ethylenediaminetetraacetic acid administered after injury prevented both physiologic and morphologic restitution. A Ca2+-free nutrient solution administered alone after injury prevented physiologic recovery, but although narrow gaps and lack of tight junctions were found between some cells, there was near-complete epithelial cell coverage. The addition of 2 mM Ca2+ to these tissues 3 h after injury effected rapid recovery of electrophysiologic parameters and a complete closure of the intercellular spaces. Cytochalasin B (3 X 10(-3) M nutrient) prevented physiologic recovery and mucosal restitution. Neither cycloheximide nor colchicine had any effect on the normal process of restitution. Autoradiography of [3H]thymidine incorporation showed no increase in labeling within 4 h of hyperosmolar injury. We conclude that adequate Ca2+ is required for complete restitution of gastric mucosa after hyperosmolar injury, and that restitution occurs by migration of persisting viable gastric pit cells.

Restitution of frog gastric mucosa in vitro: Effect of basic fibroblast growth factor

BACKGROUND Rapid re-epithelialization after superficial gastric mucosal injury is caused by migration of persisting viable epithelial cells. Basic fibroblast growth factor (bFGF) has been reported to enhance the healing of experimental duodenal ulcer, but its mode of action is unclear. The present experiments examine whether an effect of bFGF on restitution might contribute to such healing. METHODS Paired halves of bullfrog fundic gastric mucosa in Ussing chambers were injured by luminal exposure to 1 mol/L NaCl for 10 minutes. RESULTS Luminal protamine or suramin, both known to interfere with endogenous bFGF, significantly inhibited electrophysiological recovery at neutral luminal pH (pHL). Luminal sucrose octasulfate, which prevents acid degradation of bFGF, and an exogenous, acid-resistant form of bFGF allowed electrophysiological recovery at a pHL of 3.0 that completely prevented restitution in control tissues. Electrophysiological recovery correlated well with morphological restitution. The presence of endogenous bFGF in normal and restituting bullfrog mucosa was confirmed by positive staining with a monoclonal antibody. CONCLUSIONS It is concluded that rapid epithelial repair after surface injury is at least in part mediated by bFGF.

A rapid method for culturing guinea pig gastric mucous cell monolayers.

SummaryA method has been developed for growing confluent primary cultured monolayers of guinea pig gastric mucous cells suitable for in vitro electrophysiological, transport, and pharmacological studies. Isolated mucous cells were enriched on a one-step Percoll density gradient and plated on fibronectin-coated plastic dishes or in small cups with holes containing glutaraldehyde-fixed Vitrogen gels. These cups were designed to fit in Ussing chambers. Mucous cells attached, proliferated, and formed confluent monolayers in 3 d. The low cuboidal cells contained periodic acid Schiff-positive mucous granules that were negative by Bowie and indirect immunofluorescent staining for pepsinogen. Electron microscopy revealed polarized mucous cells with microvilli, mucous granules, microfilaments, small mitochondria, some vacuoles, and junctional complexes that excluded wheat germ agglutinin-peroxidase. No basal lamina was present. Monolayers could be maintained for over 2 wk but subcultures were not made. The cultures were virtually free of fibroblasts. Epithelial sheets produced by this simple and rapid method can be used for electrophysiological, ion transport, and pharmacological studies.

Functional expression of VIP receptors in normal, immortalized and transformed mammary epithelial cells

The effect of VIP and its related peptides on cAMP production has been characterized: 1) in long term culture of normal human mammary epithelial cells (HMEC); 2) in immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40; 3) in the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1D, described to exhibit normal morphogenesis in vivo and functional differentiation in vitro. Basal cAMP levels were increased 1.5- to 8.7-fold in mammary epithelial cells (p less than 0.001-0.05), with a potency EC50 = 0.02-0.6 nM VIP. The pharmacological specificity of the VIP receptors coupled to cAMP generation was established according to the following potency sequence: VIP greater than PACAP-38 greater than helodermin greater than PHM, PHV greater than helospectin 1 much greater than hpGRF, secretin in HMEC, VIP greater than PACAP-38 greater than helodermin greater than helospectin 1, PHM, PHV greater than hpGRF greater than secretin in S1T3 cells, and VIP, PHI, helodermin greater than PHV greater than rhGRF greater than secretin in HC-11 cells. Our data demonstrate the presence of functional, highly sensitive and specific VIP receptors in normal, immortalized and transformed mammary epithelial cells, suggesting a regulatory role for this neuropeptide on the growth, differentiation and function in normal and neoplastic breast tissue.

Mechanism of the Inhibitory Action of Electric Current on H+ Secretion by Frog Gastric Mucosa

The mechanism of the inhibitory action of electric current on gastric H+ secretion was studied using isolated frog gastric mucosa. Electric current, 500 μA/cm2, passed from mucosa to serosa (mucoso positive with respect to serosa), reversibly inhibited net H+ secretion, leading to complete cessation of luminal H+ appearance usually within 1 hr. The decrease in luminal H+ appearance was associated with an almost equal decrease in serosal HCO3− appearance. This is consistent with the suggestion that the inhibition of net H+ secretion was due to decrease in active H+ transport rather than to passive electrochemical back flux of the secreted H+ from the luminal bathing solution. Ultrastructural analysis of the tissues supported this view by demonstrating conversion of oxyntic cells into the resting (nonsecreting) morphologic configuration during the passage of current. Blocking of the conductive pathways for K+ in the nutrient (serosal) cell membrane by adding Ba++ to the serosal bathing solution significantly opposed the inhibitory action of electric current on H+ secretion. When electric current was passed through a mucosal solution containing a high concentration of K+, slight stimulation rather than inhibition of active H+ transport occurred (as judged from serosal HCO3− appearance). In this circumstance, the oxyntic cells retained their active secretory configuration despite the opposing current. The data indicate that electric current, passed from mucosa to serosa, inhibits active H+ transport by oxyntic cells. The data also suggest that the inhibitory action of an opposing current may be accomplished by electrolytic removal of K+ from an intracellular compartment essential for the function of the H+ secretory pump.