Integrity of the occluding barrier in high-resistant thyroid follicular epithelium in culture. I. Dependence of extracellular Ca2+ is polarized.
The dependence of the thyroid occluding barrier on extracellular Ca2+ (Ca2+e) was studied in porcine thyrocyte monolayers resting on the collagen-coated porous filter of bicameral culture chambers. Ca2+ in the apical and basal media was selectively removed by ethylene glycol-bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), and the effect on occluding junction integrity was estimated by measuring the transepithelial electrical resistance (RTE) and the transepithelial flux (FTE) of 125I-, [3H]inulin and 125I-thyroglobulin. Experiments were also carried out to monitor the recovery of the occluding barrier after restitution of Ca2+e as well as to examine the effect of a second Ca2+ depletion period. Obtained data were correlated to changes in thyrocyte ultrastructure and distribution of filamentous actin as visualized by fluorescein isothiocyanate (FITC)-phalloidin. The thyrocyte monolayer developed RTE greater than 6000 ohms.cm2 after 6 to 8 days of culture. Total depletion of Ca2+e caused a rapid fall in RTE to about 50 ohms.cm2 after 10 min. Selective removal of Ca2+ in the basal medium induced a similar decline in RTE, which, however, was significantly retarded. Removal of apical Ca2+e alone was without effect on RTE. Corresponding responses were found in FTE experiments: the apicobasal transfer of radiotracers was unaltered after apical depletion, intermediary increased after basal depletion and maximally increased after total depletion of Ca2+e. Morphologically, monolayers lacking apical Ca2+e appeared normal except for shortening of microvilli at the apical plasma membrane. Removal of basal Ca2+e caused a focal dissociation of the adherens junction, which was most pronounced at the three-cell corners, leaving a slight dilation of the apical-most portion of the intercellular cleft. The adherent junction-associated actin bundle was partly retracted into the apical cytoplasm. The former occluding junction was distended and of reduced height at the sites of dissociated adherens junction. After total Ca2+ depletion, more severe alterations were found, i.e., contraction of cells and marked dilation of intercellular clefts. Readdition of Ca2+ to cultures depleted of basal Ca2+e for 10 min normalized RTE, FTE, and junctional morphology within 30 to 60 min. When a second period of basal depletion was introduced after recovery was obtained, the decrease in RTE and the retraction of the actin bundle occurred much more rapidly than during the first depletion. It is concluded that the thyroid occluding barrier depends on extracellular Ca2+ present only at the basal side of the occluding junction. Furthermore, this Ca2+ dependence seems to be spatially segregated along the lateral plasma membrane as well as along the circumference of the thyrocyte.(ABSTRACT TRUNCATED AT 400 WORDS)
Nilsson
M
M
Department of Anatomy, University of Göteborg, Sweden.
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D004553:Electric Conductivity / Q000187:drug effects; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes; D007365:Intercellular Junctions / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010590:Phalloidine / Q000378:metabolism; D013552:Swine; D013961:Thyroid Gland / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D013972:Thyrotropin / Q000494:pharmacology
D005456:Fluorescent Dyes; D010590:Phalloidine; D013972:Thyrotropin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium