Cellular differentiation is required for cAMP but not Ca(2+)-dependent Cl- secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator
The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated &quo...
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Published in | The Journal of biological chemistry Vol. 267; no. 8; pp. 5575 - 5583 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
15.03.1992
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Subjects | |
Online Access | Get full text |
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Summary: | The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial
cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored.
Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates
in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion
in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines
were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined
by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the
unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the
unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist,
stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days
post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled
by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane
patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed
in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with
the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these
studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's
ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular
polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation
are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization
but was reduced when the cells formed tight junctions. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)42804-9 |